Polypeptides

ABSTRACT

The present disclosure relates to a class of engineered polypeptides having a binding affinity for albumin. It also relates to new methods and uses that exploit binding by these and other compounds to albumin in different contexts, some of which have significance for treatment or diagnosis of disease in mammals including humans.

TECHNICAL FIELD

The present disclosure relates to a class of engineered polypeptideshaving a binding affinity for albumin. It also relates to new methodsand uses that exploit binding by these and other compounds to albumin indifferent contexts, some of which have significance for the treatment ofdisease in mammals including humans.

BACKGROUND Serum Albumin

Serum albumin is the most abundant protein in mammalian sera (40 g/l;approximately 0.7 mM in humans), and one of its functions is to bindmolecules such as lipids and bilirubin (Peters, Advances in ProteinChemistry 37:161, 1985). Serum albumin is devoid of any enzymatic orimmunological function. Furthermore, human serum albumin (HSA) is anatural carrier involved in the endogenous transport and delivery ofnumerous natural as well as therapeutic molecules (Sellers andKoch-Weser, Albumin Structure, Function and Uses, eds Rosenoer et al,Pergamon, Oxford, p 159, 1977). The half life of serum albumin isdirectly proportional to the size of the animal, where for example humanserum albumin has a half life of 19 days and rabbit serum albumin has ahalf life of about 5 days (McCurdy et al, J Lab Clin Med 143:115, 2004).HSA is widely distributed throughout the body, in particular in theinterstitial and blood compartments, where it is mainly involved in themaintenance of osmolarity. Structurally, albumins are single-chainproteins comprising three homologous domains and in total 584 or 585amino acids (Dugaiczyk et al, Proc Natl Acad Sci USA 79:71, 1982).Albumins contain 17 disulfide bridges and a single reactive thiol,cysteine in position 34, but lack N-linked and O-linked carbohydratemoieties (Peters, 1985, supra; Nicholson et al, Br J Anaesth 85:599,2000).

Fusion or Association with HSA Results in Increased In Vivo Half Life ofProteins

Several strategies have been reported to either covalently coupleproteins directly to serum albumins or to a peptide or protein that willallow in vivo association to serum albumins. Examples of the latterapproach have been described e.g. in WO91/01743, in WO01/45746 and inDennis et al (J Biol Chem 277:35035-43, 2002). The first documentdescribes inter alia the use of albumin binding peptides or proteinsderived from streptococcal protein G (SpG) for increasing the half lifeof other proteins. The idea is to fuse the bacterially derived, albuminbinding peptide/protein to a therapeutically interestingpeptide/protein, which has been shown to have a rapid elimination fromblood. The thus generated fusion protein binds to serum albumin in vivo,and benefits from its longer half life, which increases the net halflife of the fused therapeutically interesting peptide/protein.WO01/45746 and Dennis et al relate to the same concept, but here, theauthors utilize relatively short peptides to bind serum albumin. Thepeptides were selected from a phage displayed peptide library. In Denniset al, earlier work is mentioned in which the enhancement of animmunological response to a recombinant fusion of the albumin bindingdomain of streptococcal protein G to human complement receptor Type 1was found. US patent application published as US2004/0001827 (Dennis)also discloses the use of constructs comprising peptide ligands, againidentified by phage display technology, which bind to serum albumin andwhich are conjugated to bioactive compounds for tumor targeting.

Albumin Binding Domains of Bacterial Receptor Proteins

Streptococcal protein G (SpG) is a bi-functional receptor present on thesurface of certain strains of streptococci and is capable of binding toboth IgG and serum albumin (Björck et al, Mol Immunol 24:1113, 1987).The structure is highly repetitive with several structurally andfunctionally different domains (Guss et al, EMBO J 5:1567, 1986), moreprecisely three Ig-binding domains and three serum albumin bindingdomains (Olsson et al, Eur J Biochem 168:319, 1987). The structure ofone of the three serum albumin binding domains in SpG has beendetermined, showing a three-helix bundle fold (Kraulis et al, FEBS Lett378:190, 1996, Johansson et al, J. Biol. Chem. 277:8114-20, 2002). A 46amino acid motif was defined as ABD (albumin binding domain) and hassubsequently also been designated G148-GA3 (GA for protein G-relatedalbumin binding). In for example WO09/016,043, albumin binding variantsof the 46 amino acid motif ABD are disclosed.

Other bacterial albumin binding domains than the ones in protein G havealso been identified, some of which are structurally similar to the onesof protein G. Examples of proteins containing such albumin bindingdomains are the PAB, PPL, MAG and ZAG proteins (Rozak et al,Biochemistry 45:3263-3271, 2006). Studies of structure and function ofsuch albumin binding domains have been carried out and reported e.g. byJohansson and co-workers (Johansson et al, J Mol Biol 266:859-865,1997). Furthermore, Rozak et al have reported on the creation ofartificial variants of G148-GA3, which were selected and studied withregard to different species specificity and stability (Rozak et al,Biochemistry 45:3263-3271, 2006), whereas Jonsson et al developedartificial variants of G148-GA3 having very much improved affinity forhuman serum albumin (Jonsson et al, Prot Eng Des Sel 21:515-27, 2008).For some of the variants a higher affinity was achieved at the cost ofreduced thermal stability.

In addition to the three-helix containing proteins described above,there are also other unrelated bacterial proteins that bind albumin.

ABD and Immunization

Recently, a few T- and B-cell epitopes were experimentally identifiedwithin the albumin binding region of Streptococcal protein G strain 148(G148) (Goetsch et al, Clin Diagn Lab Immunol 10:125-32, 2003). Theauthors behind the study were interested in utilizing the T-cellepitopes of G148 in vaccines, i.e. to utilize the inherentimmune-stimulatory property of the albumin binding region. Goetsch et aladditionally found a B-cell epitope, i.e. a region bound by antibodiesafter immunization, in the sequence of G148.

In pharmaceutical compositions for human administration noimmune-response is desired. Therefore, the albumin binding domain G148is as such unsuitable for use in such compositions due to itsabovementioned immune-stimulatory properties.

DESCRIPTION

The above drawbacks and deficiencies of the prior art are overcome oralleviated by, in a first aspect, an albumin binding polypeptide,comprising an amino acid sequence selected from

i)  LAX₃AKX₆X₇ANX₁₀ ELDX₁₄YGVSDF YKRLIX₂₆KAKT  VEGVEALKX₃₉X₄₀ ILX₄₃X₄₄LPwherein independently of each otherX₃ is selected from E, S, Q and C;X₆ is selected from E, S and C;X₇ is selected from A and S;X₁₀ is selected from A, S and R;X₁₄ is selected from A, S, C and K;X₂₆ is selected from D and E;X₃₉ is selected from D and E;X₄₀ is selected from A and E;X₄₃ is selected from A and K;X₄₄ is selected from A, S and E;L in position 45 is present or absent; andP in position 46 is present or absent;andii) an amino acid sequence which has at least 95% identity to thesequence defined in i).

The above defined class of sequence related polypeptides having abinding affinity for albumin is derived from a common parent polypeptidesequence, which folds into a three alpha helix bundle domain. Morespecifically, the polypeptides as described above are derived from amodel building based on a structure of a complex between serum albuminand the albumin binding domain G148-GA3 (Lejon et al, J Biol Chem279:42924-8, 2004), as well as analyses of binding and structuralproperties of a number of mutational variants of the common parentpolypeptide sequence. The above defined amino acid sequence i) comprisesamino acid substitutions as compared to the parent polypeptide sequencethat result in a class of polypeptides which are expected to fold intoan almost identical three helix bundle domain. While the parentpolypeptide sequence already comprises a binding surface for interactionwith albumin, that binding surface is modified by some of thesubstitutions according to the above definition. The substitutionsaccording to the above definition provide an improved albumin bindingability as compared to the parent polypeptide sequence.

The albumin binding polypeptides according to the first aspect of thedisclosure exhibit a set of characteristics, which, for example, makethem suitable for use as fusion or conjugate partners for therapeuticmolecules for human administration. The albumin binding polypeptidesaccording to the present disclosure demonstrate, for example incomparison with related albumin binding polypeptides such as the albuminbinding domain G148-GA3 and the albumin binding polypeptides disclosedin WO09/016,043, at least five of the following six characteristics:

-   -   The polypeptides display a different surface compared to, for        example, G148-GA3 and other bacterially derived albumin binding        domains. The difference may decrease or eliminate any risk for        antibody reactions in a subject, such as a human, which has been        previously exposed to such bacterial proteins.    -   The polypeptides comprise fewer potential T-cell epitopes than,        for example, G148-GA3 and other related, but different,        mutational variants of the common parent polypeptide sequence,        and hence exhibit low immunogenicity when administered to a        subject, such as a human.    -   The polypeptides display a lower reactivity with circulating        antibodies when administered to a subject, such as a human.        Thus, by amino acid substitutions in the surface of the        polypeptides exposed to circulating antibodies, i.e. in the        polypeptide surface not involved in the binding interaction with        albumin, antibody cross-reactivity is reduced as compared to,        for example, antibody cross-reactivity caused by G148-GA3 as        measured in a test set of human sera.    -   The polypeptides have a high albumin binding ability, both in        terms of a higher binding affinity, as defined by a K_(D) value,        and in terms of a slower off-rate, as defined by a k_(off)        value, than, for example, known naturally occurring albumin        binding polypeptides, such as the albumin binding domains        derived from bacterial proteins.    -   The polypeptides comprise fewer amino acid residues that are        associated with stability problems of polypeptides than, for        example, known naturally occurring albumin binding polypeptides,        such as the albumin binding domains derived from bacterial        proteins. Thus, the polypeptides comprise, for example, no        oxidation-prone methionines or tryptophanes and only one        asparagine.    -   The polypeptides have a higher structural stability, as defined        by a melting point of above 55° C., than previous albumin        binding polypeptides, such as those disclosed in WO09/016,043.

In one embodiment, the albumin binding polypeptide according to thefirst aspect display all six of the above listed characteristics. Inanother embodiment, the albumin binding polypeptide according to thefirst aspect displays, when bound to albumin, a more hydrophilic profilethan, for example, previous albumin binding polypeptides, such as thosedisclosed in WO09/016,043. The surface of the albumin bindingpolypeptide which is exposed to the surroundings when the polypeptideinteracts with albumin comprises fewer amino acid residues that confersurface hydrophobicity.

As the skilled person will realize, the function of any polypeptide,such as the albumin binding capacity of the polypeptides according tothe first aspect, is dependent on the tertiary structure of thepolypeptide. It is however possible to make changes to the sequence ofamino acids in an α-helical polypeptide without affecting the structurethereof (Taverna and Goldstein, J Mol Biol 315(3):479-84, 2002; He etal, Proc Natl Acad Sci USA 105(38):14412-17, 2008). Thus, modifiedvariants of i), which are such that the resulting sequence is at least95% identical to a sequence belonging to the class defined by i), arealso encompassed by the first aspect. For example, it is possible thatan amino acid residue belonging to a certain functional grouping ofamino acid residues (e.g. hydrophobic, hydrophilic, polar etc) could beexchanged for another amino acid residue from the same functional group.

The term “% identical” or “% identity”, as used in the specification andclaims, is calculated as follows. The query sequence is aligned to thetarget sequence using the CLUSTAL W algorithm (Thompson, J. D., Higgins,D. G. and Gibson, T. J., Nucleic Acids Research, 22: 4673-4680 (1994)).A comparison is made over the window corresponding to the shortest ofthe aligned sequences. The shortest of the aligned sequences may in someinstances be the target sequence, such as the albumin bindingpolypeptide disclosed herein. In other instances, the query sequence mayconstitute the shortest of the aligned sequences. The query sequence mayfor example consist of at least 10 amino acid residues, such as at least20 amino acid residues, such as at least 30 amino acid residues, such asat least 40 amino acid residues, for example 45 amino acid residues. Theamino acid residues at each position are compared, and the percentage ofpositions in the query sequence that have identical correspondences inthe target sequence is reported as % identity.

In one embodiment of the albumin binding polypeptide according to thefirst aspect, X₆ is E.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₃ is S.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₃ is E.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₇ is A.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₁₄ is S.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₁₄ is C.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₁₀ is A.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₁₀ is S.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₂₆ is D.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₂₆ is E.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₃₉ is D.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₃₉ is E.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₄₀ is A.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₄₃ is A.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₄₄ is A.

In another embodiment of the albumin binding polypeptide according tothis aspect, X₄₄ is S.

In another embodiment of the albumin binding polypeptide according tothis aspect, the L residue in position 45 is present.

In another embodiment of the albumin binding polypeptide according tothis aspect, the P residue in position 46 is present.

In another embodiment of the albumin binding polypeptide according tothis aspect, the P residue in position 46 is absent.

In another embodiment, the albumin binding polypeptide according to thisaspect is subject to the proviso that X₇ is neither L, E nor D.

The albumin binding polypeptide according to the first aspect may beprepared for conjugation with a suitable conjugation partner by thereplacement of surface exposed amino acid residues with, for example,either a cysteine or a lysine. These replacements may be introduced intothe N-terminal helix, i.e. helix one, of the polypeptide, which is thehelix situated furthest away from the serum albumin when the albuminbinding polypeptide is bound to serum albumin. Thus, a lysine residue inposition X₁₄ of the sequence defined in i) may be used to enablesite-directed conjugation. This may furthermore be advantageous when themolecule is made by chemical peptide synthesis, since orthogonalprotection of the epsilon-amino group of said lysine may be utilized.Furthermore, a cysteine residue may be introduced into the amino acidsequence to enable site-directed conjugation. For example, a cysteineresidue may be introduced into any one of the positions X₃, X₆ and/orX₁₄ in accordance with the above definition.

Coupling of a conjugation partner to the epsilon-amine of a lysine orthe thiol group of a cysteine represents two chemically differentalternatives to obtain site-directed conjugation using an amino acidresidue within the amino acid sequence i). As the skilled personunderstands, other chemical alternatives for preparing an amino acidsequence for conjugation exist, and are as such also within the scope ofthe present disclosure. One example of such a chemistry is theclick-like chemistry enabled by the introduction of a tyrosine aspresented by Ban et al (J Am Chem Soc 132:1523-5, 2009).

The terms “albumin binding” and “binding affinity for albumin” as usedin this specification refer to a property of a polypeptide which may betested for example by the use of surface plasmon resonance technology,such as in a Biacore instrument. For example as described in theexamples below, albumin binding affinity may be tested in an experimentin which albumin, or a fragment thereof, is immobilized on a sensor chipof the instrument, and the sample containing the polypeptide to betested is passed over the chip. Alternatively, the polypeptide to betested is immobilized on a sensor chip of the instrument, and a samplecontaining albumin, or a fragment thereof, is passed over the chip.Albumin may, in this regard, be a serum albumin from a mammal, such ashuman serum albumin. The skilled person may then interpret the resultsobtained by such experiments to establish at least a qualitative measureof the binding affinity of the polypeptide for albumin. If aquantitative measure is desired, for example to determine a K_(D) valuefor the interaction, surface plasmon resonance methods may also be used.Binding values may for example be defined in a Biacore2000 instrument(GE Healthcare). Albumin is suitably immobilized on a sensor chip of themeasurement, and samples of the polypeptide whose affinity is to bedetermined are prepared by serial dilution and injected. K_(D) valuesmay then be calculated from the results using for example the 1:1Langmuir binding model of the BIAevaluation 4.1 software provided by theinstrument manufacturer (GE Healthcare).

In one embodiment, the albumin binding polypeptide according to thisaspect binds to albumin such that the k_(off) value of the interactionis at most 5×10⁻⁵ s⁻¹, such as at most 5×10⁻⁶ s⁻¹.

As described above, the albumin binding polypeptides as defined by theamino acid sequence i) are derived from a common parent polypeptidesequence which folds into a three alpha helix bundle domain. In oneembodiment, the three helix domain of this parent polypeptide sequenceforms part of protein G from Streptococcus strain G148, in particulardomain GA3.

In another embodiment, the amino acid sequence of the albumin bindingpolypeptide is selected from any one of SEQ ID NO:1-144 and SEQ IDNO:164-203, such as selected from any one of SEQ ID NO:1-144. Morespecifically, the amino acid sequence is selected from SEQ ID NO:4-5,SEQ ID NO:7-8, SEQ ID NO:10-11, SEQ ID NO:13-14, SEQ ID NO:16-17, SEQ IDNO:19-20, SEQ ID NO:22-23, SEQ ID NO:25-26, SEQ ID NO:28-29, SEQ IDNO:31-32, SEQ ID NO:34-35, SEQ ID NO:37-38, SEQ ID NO:41-42, SEQ IDNO:49-50, SEQ ID NO:164-170 and SEQ ID NO:192-203. Thus, the amino acidsequence may be selected from SEQ ID NO:4-5, SEQ ID NO:7-8, SEQ IDNO:10-11, SEQ ID NO:13-14, SEQ ID NO:16-17, SEQ ID NO:19-20, SEQ IDNO:22-23, SEQ ID NO:25-26, SEQ ID NO:28-29, SEQ ID NO:31-32, SEQ IDNO:34-35, SEQ ID NO:37-38, SEQ ID NO:41-42 and SEQ ID NO:49-50.

In one embodiment, the albumin binding polypeptide according to thisaspect further comprises one or more additional amino acid residuespositioned at the N- and/or the C-terminal of the sequence defined ini). These additional amino acid residues may play a role in enhancingthe binding of albumin by the polypeptide, and improving theconformational stability of the folded albumin binding domain, but mayequally well serve other purposes, related for example to one or more ofproduction, purification, stabilization in vivo or in vitro, coupling,labeling or detection of the polypeptide, as well as any combinationthereof. Such additional amino acid residues may comprise one or moreamino acid residue(s) added for purposes of chemical coupling, e.g. to achromatographic resin to obtain an affinity matrix or to a chelatingmoiety for complexing with a radiometal.

The amino acids directly preceding or following the alpha helix at theN- or C-terminus of the amino acid sequence i) may thus in oneembodiment affect the conformational stability. One example of an aminoacid residue which may contribute to improved conformational stabilityis a serine residue positioned at the N-terminal of the amino acidsequence i) as defined above. The N-terminal serine residue may in somecases form a canonical S-X-X-E capping box, by involving hydrogenbonding between the gamma oxygen of the serine side chain and thepolypeptide backbone NH of the glutamic acid residue. This N-terminalcapping may contribute to stabilization of the first alpha helix of thethree helix domain constituting the albumin binding polypeptideaccording to the first aspect of the disclosure.

Thus, in one embodiment, the additional amino acids comprise at leastone serine residue at the N-terminal of the polypeptide. The amino acidsequence is in other words preceded by one or more serine residue(s). Inanother embodiment of the albumin binding polypeptide, the additionalamino acids comprise a glycine residue at the N-terminal of thepolypeptide. It is understood that the amino acid sequence i) may bepreceded by one, two, three, four or any suitable number of amino acidresidues. Thus, the amino acid sequence may be preceded by a singleserine residue, a single glycine residue or a combination of the two,such as a glycine-serine (GS) combination or a glycine-serine-serine(GSS) combination. Examples of albumin binding polypeptides comprisingadditional amino residues at the N-terminal are set out in SEQ IDNO:145-163, such as in SEQ ID NO:145-148 and SEQ ID NO:162-163. In yetanother embodiment, the additional amino acid residues comprise aglutamic acid at the N-terminal of the polypeptide as defined by thesequence i).

Similarly, C-terminal capping may be exploited to improve stability ofthe third alpha helix of the three helix domain constituting the albuminbinding polypeptide. A proline residue, when present at the C-terminalof the amino acid sequence defined in i), may at least partly functionas a capping residue. In such a case, a lysine residue following theproline residue at the C-terminal may contribute to furtherstabilization of the third helix of the albumin binding polypeptide, byhydrogen bonding between the epsilon amino group of the lysine residueand the carbonyl groups of the amino acids located two and threeresidues before the lysine in the polypeptide backbone, e.g., when bothL45 and P46 are present, the carbonyl groups of the leucine and alanineresidues of the amino acid sequence defined in i). Thus, in oneembodiment, the additional amino acids comprise a lysine residue at theC-terminal of the polypeptide.

As discussed above, the additional amino acids may be related to theproduction of the albumin binding polypeptide. In particular, when analbumin binding polypeptide according to an embodiment in which P46 ispresent is produced by chemical peptide synthesis, one or more optionalamino acid residues following the C-terminal proline may provideadvantages. Such additional amino acid residues may for example preventformation of undesired substances, such as diketopiperazine at thedipeptide stage of the synthesis. One example of such an amino acidresidue is glycine. Thus, in one embodiment, the additional amino acidscomprise a glycine residue at the C-terminal of the polypeptide,directly following the proline residue or following an additional lysineand/or glycine residue as accounted for above. Alternatively,polypeptide production may benefit from amidation of the C-terminalproline residue of the amino acid sequence i), when present. In thiscase, the C-terminal proline comprises an additional amine group at thecarboxyl carbon. In one embodiment of the polypeptides described herein,particularly those ending at its C-terminus with proline or other aminoacid known to racemize during peptide synthesis, the above-mentionedaddition of a glycine to the C-terminus or amidation of the proline,when present, can also counter potential problems with racemization ofthe C-terminal amino acid residue. If the polypeptide, amidated in thisway, is intended to be produced by recombinant means, rather than bychemical synthesis, amidation of the C-terminal amino acid can beperformed by several methods known in the art, e.g. through the use ofamidating PAM enzyme.

Examples of albumin binding polypeptides comprising additional aminoacid residues at the C-terminal are set out in SEQ ID NO:145-152, suchas in SEQ ID NO:148-150. The skilled person is aware of methods foraccomplishing C-terminal modification, such as by different types ofpre-made matrices for peptide synthesis.

In another embodiment, the additional amino acid residues comprise acysteine residue at the N- and/or C-terminal of the polypeptide. Such acysteine residue may directly precede and/or follow the amino acidsequence as defined in i) or may precede and/or follow any otheradditional amino acid residues as described above. Examples of albuminbinding polypeptides comprising a cysteine residue at the N- and/orC-terminal of the polypeptide chain are set out in SEQ ID NO:149-150(C-terminal) and SEQ ID NO:151-152 (N-terminal). By the addition of acysteine residue to the polypeptide chain, a thiol group for sitedirected conjugation of the albumin binding polypeptide may be obtained.Alternatively, a selenocysteine residue may be introduced at theC-terminal of the polypeptide chain, in a similar fashion as for theintroduction of a cysteine residue, to facilitate site-specificconjugation (Cheng et al, Nat Prot 1:2, 2006).

In one embodiment, the albumin binding polypeptide comprises no morethan two cysteine residues. In another embodiment, the albumin bindingpolypeptide comprises no more than one cysteine residue.

In another embodiment, the additional amino acid residues of the albuminbinding polypeptide comprise a “tag” for purification or detection ofthe polypeptide, such as a hexahistidyl (His₆) tag, or a “myc” (“c-Myc”)tag or a “FLAG” tag for interaction with antibodies specific to the tagand/or to be used in purification. The skilled person is aware of otheralternatives.

In yet another embodiment, the albumin binding polypeptide according tothis aspect binds to human serum albumin. In other embodiments, thealbumin binding polypeptide binds to albumin from other species than thehuman species, such as albumin from mouse, rat, dog and cynomolgusmacaques.

The “additional amino acid residues” discussed above may also constituteone or more polypeptide domain(s) with any desired function, such as thesame binding function as the first, albumin binding domain, or anotherbinding function, or a therapeutic function, or an enzymatic function,or a fluorescent function, or mixtures thereof.

There is consequently in another, related aspect, provided a fusionprotein or conjugate comprising

-   i) a first moiety consisting of an albumin binding polypeptide    according to the first aspect as described herein; and-   ii) a second moiety consisting of a polypeptide having a desired    biological activity.

A fusion protein or conjugate comprising an albumin binding polypeptideaccording to the first aspect of the disclosure and a second moiety mayincrease the in vitro and/or the in vivo half life of the second moiety,as compared to the in vivo half life of the second moiety per se. As aconsequence, when a fusion protein or conjugate according to this aspectis administered to a subject, such as a human subject, the in vivoexposure to the second moiety may increase, which may lead to improvedpotency of the biological activity of the second moiety, as compared tothe potency of in vivo exposure of the second moiety when administeredby itself.

The desired biological activity may, for example, be a therapeuticactivity, a binding activity or an enzymatic activity. When the desiredbiological activity is a therapeutic activity, the second moiety showingthis activity may be a therapeutically active polypeptide. Non-limitingexamples of therapeutically active polypeptides are biomolecules, suchas molecules selected from the group consisting of human endogenousenzymes, hormones, growth factors, chemokines, cytokines andlymphokines, and non-human biologically active proteins, such asproteins selected from the group consisting of bacterial toxins (e.g.pseudomonas exotoxin and staphylococcal and streptococcalsuperantigens), enzymes (e.g. RNase and beta-lactamase) and activatingproteins (e.g. streptokinase). Non-limiting examples of therapeuticallyactive biomolecules which may prove useful in a fusion or conjugate withthe albumin binding polypeptide are selected from the group consistingof IL-2, GLP-1, BNP (Alb-beta-natriuretic peptide), IL-1-RA(interleukin-1 receptor antagonist), KGF (keratinocyte growth factor),Stemgen®, growth hormone (GH), G-CSF, CTLA-4, myostatin, Factor VII,Factor VIII and Factor IX.

The leaky defective blood vessels of tumor tissue make its vasculature(endothelial barrier) permeable for macromolecules, whereas in bloodvessels of healthy tissue only small molecules can pass the endothelialbarrier. Likewise, the permeability of the blood-joint barrier foralbumin in inflamed joints of rheumatoid arthritis patients is markedlyincreased. Thus, fusion proteins or conjugates according to this aspectare likely to permeate blood vessels in tumor tissue and the blood-jointbarrier in inflamed joints.

When said desired biological activity of the second moiety is a bindingactivity, said second moiety may be a binding polypeptide capable ofselective interaction with a target molecule. Such a binding polypeptidemay for example be selected from the group consisting of antibodies andfragments and domains thereof substantially retaining antibody bindingactivity; microbodies, maxybodies, avimers and other smalldisulfide-bonded proteins; and binding proteins derived from a scaffoldselected from the group consisting of staphylococcal protein A anddomains thereof, other three helix domains, lipocalins, ankyrin repeatdomains, cellulose binding domains, γ crystallines, green fluorescentprotein, human cytotoxic T lymphocyte-associated antigen 4, proteaseinhibitors such as Kunitz domains, PDZ domains, SH3 domains, peptideaptamers, staphylococcal nuclease, tendamistats, fibronectin type IIIdomain, transferrin, zinc fingers and conotoxins.

In some embodiments, the target molecule for binding of said targetbinding polypeptide may be selected from the group consisting of amyloidβ (Aβ) peptide of Alzheimer's disease; other disease-associated amyloidpeptides; toxins, such as bacterial toxins and snake venoms; bloodclotting factors, such as von Willebrand factor; interleukins, such asIL-13; myostatin; pro-inflammatory factors, such as TNF-α, TNF-αreceptor, IL-1, IL-8 and IL-23; complement factors, such as C3 and C5;hypersensitivity mediators, such as histamine and IgE; tumor-relatedantigens, such as CD19, CD20, CD22, CD30, CD33, CD40, CD52, CD70, cMet,HER1, HER2, HER3, HER4, CAIX (carbonic anhydrase IX), CEA, IL-2receptor, MUC1, PSMA, TAG-72; and other biological molecules such asG-CSF, GM-CSF, growth hormone (GH), insulin and somatostatin.

As the skilled person understands, the albumin binding polypeptideaccording to the first aspect may be useful in a fusion protein or as aconjugate partner to any other moiety. Therefore, the above lists oftherapeutically active polypeptides, binding polypeptides and targetmolecules should not be construed as limiting in any way.

In one embodiment of a fusion protein or conjugate according to thepresent disclosure, the second moiety is conjugated to the albuminbinding polypeptide via a lysine or cysteine residue added to the N- orC-terminal of the albumin binding polypeptide or via a lysine orcysteine residue at a position within the albumin binding polypeptide,such as at a position selected from X₃, X₆ and X₁₄. If the conjugationsite is one within the amino acid sequence i) of the albumin bindingpolypeptide, such as a cysteine in position X₁₄, no additional aminoacids need to be added to the albumin binding polypeptide for thepurpose of enabling conjugation to the second moiety. A conjugation sitewithin the polypeptide chain as defined by i) may moreover shield thepolypeptide against cross-reacting antibodies, in particular the portionof the polypeptide close to the conjugation site. Without wishing to bebound by theory, when the conjugate via the albumin binding polypeptideis bound to serum albumin in vivo, i.e. situated in the binding pocketof serum albumin, the second moiety, conjugated at a position within forexample helix one of the three helix domain constituting the albuminbinding polypeptide, is likely to point away from the serum albumin towhich the albumin binding polypeptide is bound. In addition, aconjugation site within the albumin binding polypeptide may impair thepresentation of the portion of the peptides otherwise derived from thisportion of the polypeptide to T-cells due to, for example, effects onprocessing in the antigen presenting cell, impaired fit of potentialpeptides in the peptide binding grove of the MCH class II molecule, andremodeled peptide surface available to the T-cell receptor (due to theconjugated portion sticking out). Thus, the immunogenicity of theportion of the conjugate near the conjugation site is expected to becomefurther reduced after conjugation.

Due to the high affinity between the albumin binding polypeptide of thepresent disclosure and serum albumin, a conjugate or fusion proteincomprising such an albumin binding polypeptide might be regarded as anindirect complex with serum albumin. A conjugate or a fusion proteinaccording to the present disclosure thus provides an alternative to thefrequently used method of exploiting direct conjugates or fusions withserum albumin. Such direct conjugates with serum albumin are frequentlyinhomogeneous, irrespective of what method is used for coupling. When aspecific molecule is coupled to serum albumin via an amino group of alysine residue, any one of a large number of lysines on the surface ofthe serum albumin molecule may be targeted, which gives a randomconjugation site and a random product. Although thiol coupling via thesingle unpaired cysteine in human serum albumin (in position 34, Peters,1985, supra) seems to offer an alternative method for obtaining a directconjugate, such a methodology frequently does not lead to a homogeneousproduct. Only 20-60% of the molecules in commercially available (human)serum albumin display a free thiol group, whereas the rest are blockedby thiol compounds such as cysteine, homocysteine or glutathione. Incontrast, conjugation to the three helix domain of the albumin bindingpolypeptide according to the present disclosure may be performedsite-specifically. This may be accomplished, as discussed above, eitherby coupling to one or more cysteines, to a selenocysteine, or to adesignated lysine (orthogonally protected during synthesis).

According to this aspect of the present disclosure, the second moietyhaving the desired biological activity may either be conjugated to thethree helix domain of the albumin binding polypeptide or produced as afusion protein with the same. A non-limiting example of a conjugateaccording to the present disclosure is given below. Glucagon-likepeptide 1 (GLP-1), or a derivative thereof, is a small polypeptide whichmay suitably be present as a second moiety in a conjugate with thealbumin binding polypeptide. Conjugation of GLP-1 to the albumin bindingpolypeptide may be performed in any one of the positions of thepolypeptide sequence as described above. The conjugate may as such beproduced in a non-biological process and is expected to display asignificantly enhanced potency as compared to the potency of GLP-1 perse. Conjugation may be employed with both small polypeptides orproteins, such as GLP-1, or with larger polypeptides or proteins. Aconjugate according to the present disclosure may typically comprise anon-amino acid spacer moiety, such as polyethylene glycol (PEG).

Other polypeptides or proteins may be combined with the amino acidsequence of the albumin binding polypeptide in the form of a fusionprotein. Such a fusion protein may furthermore comprise one or morespacer amino acid residues between the first and second moieties.

As described above, the albumin binding polypeptide according to thefirst aspect binds serum albumin from several species, including mouse,rat, dog and cynomolgus macaques. Thus, a fusion protein or conjugateaccording to the present disclosure may contribute to enhancing thebiological effect of a second moiety, not only in a human subject, butalso in animal models. A number of endogenous proteins have beenproduced as direct fusions with human serum albumin, examples of suchproteins include G-CSF, GH, interferons, CD4, IL-2, insulin, glucagon,GLP-1, antibody Fab fragments and protease inhibitors like Kunitz-domainderived proteins. Such direct fusions may however not be fully evaluatedin animal models. This is due to the fact that human serum albumin doesnot interact properly with the endogenous Fc neonatal receptor (FcRn),e.g. in the commonly used animal models mouse and rat, and that thisinteraction is an important factor contributing to the long circulationtime of serum albumin. As described above, a conjugate or a fusionprotein according to the present disclosure may, in the presence ofserum albumin, combine with albumin and function as an indirect fusionwith albumin. This makes a conjugate or a fusion protein comprising analbumin binding polypeptide according to the first aspect useful inpreclinical model species, provided that the second moiety isbiologically active in the selected species.

In one embodiment, there is provided a fusion protein or conjugatecomprising a further moiety consisting of a polypeptide having afurther, desired biological activity, which may be the same as ordifferent from that of the second moiety. One specific example of such afusion protein or conjugate comprises a therapeutically activepolypeptide as defined above as a second moiety and a bindingpolypeptide as defined above as a further moiety.

With regard to the description above of fusion proteins or conjugatesincorporating an albumin binding polypeptide according to the firstaspect, it is to be noted that the designation of first, second andfurther moieties is made for clarity reasons to distinguish betweenalbumin binding polypeptide or polypeptides according to the presentdisclosure on the one hand, and moieties exhibiting other functions onthe other hand. These designations are not intended to refer to theactual order of the different domains in the polypeptide chain of thefusion protein or conjugate. Thus, for example, said first moiety maywithout restriction appear at the N-terminal end, in the middle, or atthe C-terminal end of the fusion protein or conjugate.

In a related aspect, there is provided an albumin binding polypeptide,fusion protein or conjugate as defined in the present disclosure,further comprising an organic molecule, such as a cytotoxic agent.Non-limiting examples of cytotoxic agents which may be fused orconjugated to an albumin binding polypeptide according to the firstaspect, or combined with a fusion protein or conjugate according to thesecond aspect, are selected from calicheamycin, auristatin, doxorubicin,maytansinoid, taxol, ecteinascidin, geldanamycin, methotrexate and theirderivatives, and combinations thereof. Previously, attempts have beenmade to treat various disorders with direct albumin conjugates. Suchdirect albumin conjugates have been exploited e.g. with doxorubicin incancer (Kratz et al, J Med Chem 45: 5523-33, 2002) and metotrexate inrheumatoid arthritis (Wunder et al, J Immunol 170:4793-4801, 2003). Itis to be understood that the albumin binding polypeptide, either byitself or as a moiety in a fusion protein or conjugate, by its highalbumin binding ability provides indirect means of construing albumincomplexes, and thus may provide an alternative treatment method comparedto the attempts mentioned above.

The above aspects furthermore encompass polypeptides in which thealbumin binding polypeptide according to the first aspect, or thealbumin binding polypeptide as comprised in a fusion protein orconjugate according to the second aspect, has been provided with a labelgroup, such as a label selected from the group consisting of fluorescentdyes and metals, chromophoric dyes, chemiluminescent compounds andbioluminescent proteins, enzymes, radionuclides and particles, forexample for purposes of detection of the polypeptide. In particular, thedisclosure encompasses a radiolabeled polypeptide consisting of aradiochelate of an albumin binding polypeptide, fusion protein orconjugate as described herein and a radionuclide, such as a radioactivemetal.

In embodiments where the labeled albumin binding polypeptide comprisesan albumin binding polypeptide according to the first aspect of thedisclosure and a label, the labeled polypeptide may for example be usedfor labeling serum albumin indirectly. Due to the strong associationbetween the labeled polypeptide and serum albumin, the labeledpolypeptide may be used for example to study vascular permeability andblood pool.

In other embodiments, the labeled albumin binding polypeptide is presentas a moiety in a fusion protein or conjugate also comprising a secondmoiety having a desired biological activity. The label may in someinstances be coupled only to the albumin binding polypeptide, and insome instances both to the albumin binding polypeptide and to the secondmoiety of the conjugate or fusion protein. When reference is made to alabeled polypeptide, this should be understood as a reference to allaspects of polypeptides as described herein, including fusion proteinsand conjugates comprising an albumin binding polypeptide and a secondand optionally further moieties. Thus, a labeled polypeptide may containonly the albumin binding polypeptide and e.g. a therapeuticradionuclide, which may be chelated or covalently coupled to the albuminbinding polypeptide, or contain the albumin binding polypeptide, atherapeutic radionuclide and a second moiety such as a small moleculehaving a desired biological activity such as therapeutic efficacy.

In embodiments where the albumin binding polypeptide, fusion protein orconjugate is radiolabeled, such a radiolabeled polypeptide may comprisea radionuclide. A majority of radionuclides have a metallic nature andmetals are typically incapable of forming stable covalent bonds withelements presented in proteins and peptides. For this reason, labelingof proteins and peptides with radioactive metals is performed with theuse of chelators, i.e. multidentate ligands, which form non-covalentcompounds, called chelates, with the metal ions. In an embodiment of thealbumin binding polypeptide, fusion protein or conjugate, theincorporation of a radionuclide is enabled through the provision of achelating environment, through which the radionuclide may becoordinated, chelated or complexed to the polypeptide.

One example of a chelator is the polyaminopolycarboxylate type ofchelator. Two classes of such polyaminopolycarboxylate chelators can bedistinguished: macrocyclic and acyclic chelators. In one embodiment, thealbumin binding polypeptide, fusion protein or conjugate comprises achelating environment provided by a polyaminopolycarboxylate chelatorconjugated to the albumin binding polypeptide via a thiol group of acysteine residue or an epsilon amine group of a lysine residue.

The most commonly used macrocyclic chelators for radioisotopes ofindium, gallium, yttrium, bismuth, radioactinides and radiolanthanidesare different derivatives of DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). In oneembodiment, the chelating environment of the albumin bindingpolypeptide, fusion protein or conjugate is provided by DOTA or aderivative thereof. More specifically, one group of chelatingpolypeptides encompassed by the present disclosure is made by reactingthe DOTA derivative 1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid-10-maleimidoethylacetamide (maleimidomonoamide-DOTA) with, forexample, a thiol group of the albumin binding polypeptide, for exampleas described in Example 5.

The high kinetic inertness, i.e. the slow rate of dissociation of metalfrom DOTA, favors stable attachment of a radionuclide. However, elevatedtemperatures are required for labeling due to a slow association rate.For this reason, DOTA derivatives are widely used for labeling of shortpeptides, such as the albumin binding polypeptides of the presentdisclosure, which display binding functionality following incubation attemperatures required for the labeling reaction.

The most commonly used acyclic polyaminopolycarboxylate chelators aredifferent derivatives of DTPA (diethylenetriamine-pentaacetic acid).Hence, polypeptides having a chelating environment provided bydiethylenetriaminepentaacetic acid or derivatives thereof are alsoencompassed by the present disclosure.

It has been found that backbone-modified semi-rigid variants of DTPAprovide adequate stability for labeling with ⁹⁰Y of e.g. Zevalin®.Though acyclic chelators are less inert, and consequently, less stablethan macrocyclic ones, their labeling is rapid enough even at ambienttemperature. For this reason, they might be used for labeling of fusionproteins or conjugates according to the present disclosure. Detailedprotocols for coupling of polyaminopolycarboxylate chelators totargeting proteins and peptides have been published by Cooper et al (NatProt 1: 314-7, 2006) and by Sosabowski and Mather (Nat Prot 1:972-6,2006).

An albumin binding polypeptide, a fusion protein or conjugate accordingto the aspects described herein coupled to a polyaminopolycarboxylatechelator may be used to provide a radiolabeled polypeptide consisting ofa radiochelate of the albumin binding polypeptide, fusion protein orconjugate coupled to the chelator and a radionuclide suitable formedical imaging, the radionuclide being selected from the groupconsisting of ⁶¹Cu, ⁶⁴Cu, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ^(110m)In, ¹¹¹In, ⁴⁴Sc and⁸⁶Y, or with a radionuclide suitable for therapy, the radionuclide beingselected from the group consisting of ²²⁵Ac, ²¹²Bi, ²¹³Bi, ⁶⁷Cu, ¹⁶⁶Ho,¹⁷⁷Lu, ²¹²Pb, ¹⁴⁹Pm, ¹⁵³Sm, ²²⁷Th and ⁹⁰Y, wherein the radionuclide iscomplexed with the albumin binding polypeptide via a chelator.

In embodiments thereof, the polypeptide may also be radiolabeled withnon-metal radioisotopes using so called indirect labeling. Thus, forlabeling with for example ¹⁸F, ⁷⁶Br, different iodine isotopes and²¹¹At, intermediate “linker molecules” are used for labeling. Such alinker molecule should contain two functional moieties, one providingrapid and efficient radiolabeling, and another enabling rapid andefficient coupling to the proteins, e.g. to amine groups, or preferablyto the thiol group of a unique cysteine, such as in position X₁₄ of thealbumin binding polypeptide. For example a malemide group reacts withthiol groups to form a stable thioether bond. The “linker molecule” mayfirst be reacted with the radiolabel and subsequently with the thiol orthe selenothiol group of the protein.

In another aspect, there is provided a polynucleotide encoding analbumin binding polypeptide or a fusion protein as described herein.Also encompassed is a method of producing an albumin binding polypeptideor a fusion protein as described above, comprising expressing thepolynucleotide, an expression vector comprising the polynucleotide and ahost cell comprising the expression vector.

The albumin binding polypeptide of the present disclosure mayalternatively be produced by non-biological peptide synthesis usingamino acids and/or amino acid derivatives having protected reactiveside-chains, the non-biological peptide synthesis comprising

step-wise coupling of the amino acids and/or the amino acid derivativesto form a polypeptide according to the first aspect having protectedreactive side-chains,

removal of the protecting groups from the reactive side-chains of thepolypeptide, and

folding of the polypeptide in aqueous solution.

Thus, normal amino acids (e.g. glycine, alanine, phenylalanine,isoleucine, leucine and valine) and pre-protected amino acid derivativesare used to sequentially build a polypeptide sequence, in solution or ona solid support in an organic solvent. One specific example of peptidesynthesis on solid support is described in Example 5. When a completepolypeptide sequence is built, the protecting groups are removed and thepolypeptide is allowed to fold in an aqueous solution. Each polypeptideaccording to the present disclosure reversibly folds into a three helixbundle domain without added factors, and hence folds spontaneously.

The conjugate according to the second aspect may be produced by a methodcomprising producing an albumin binding polypeptide according to any ofthe methods described above, such as by non-biological peptidesynthesis, and conjugating the produced albumin binding polypeptide witha second and/or further moiety as defined in connection with the secondaspect.

In one embodiment of a fusion protein or conjugate, there is moreoverprovided a fusion protein or conjugate as defined herein for use intherapy, e.g. for use as a medicament. Such a fusion protein orconjugate may exhibit a half-life in vivo which is longer than thehalf-life in vivo of the polypeptide having a desired biologicalactivity per se. The fusion protein or conjugate may moreover elicit noor a reduced immune response upon administration to the mammal, such asa human, as compared to the immune response elicited upon administrationto the mammal of the polypeptide having a desired biological activityper se. Alternatively speaking, this provides a method for decreasingthe immunogenicity of a polypeptide having a desired biologicalactivity, through the fusion or conjugation of such a polypeptide to analbumin binding polypeptide, fusion protein or conjugate according toaspects disclosed herein. In addition, this may enable enhancement ofthe biological activity of a second moiety.

In another embodiment, there is provided a fusion protein or conjugateaccording to the present disclosure, for use in diagnosis, e.g. for useas a diagnostic agent.

The present disclosure also concerns different aspects of using theabove-described albumin binding polypeptide, as well as various methodsfor treatment, diagnosis and detection in which the polypeptide isuseful due to its binding and other characteristics. When referring tothe “albumin binding polypeptide” in the following description of theseuses and methods, this term is intended to encompass the albumin bindingpolypeptide alone, but also all those molecules based on thispolypeptide described above that e.g. incorporate the albumin bindingpolypeptide as a moiety in a fusion protein or conjugate, and/or areconjugated to a label, a chelator, a therapeutic and/or diagnostic agentand/or are provided with additional amino acid residues as a tag or forother purposes. As explained above, such fusion proteins, derivativesetc form a part of the present disclosure.

Another set of aspects concern the provision of new means to increasethe solubility in aqueous solution of a poorly soluble compound, throughconjugation thereof to an albumin binding polypeptide, fusion protein orconjugate. The ensuing complex of poorly soluble compound and an albuminbinding polypeptide, alone or incorporated as a moiety in a fusionprotein or conjugate, is able to associate with albumin in vivo or invitro, which association increases the solubility in aqueous solution.Thus, in an embodiment of this further aspect, there is provided acomposition, comprising

a compound which per se has a solubility in water of no more than 100μg/ml; coupled to

an albumin binding polypeptide, a fusion protein or conjugate asdescribed herein, wherein the compound and the albumin bindingpolypeptide, fusion protein or conjugate are covalently coupled.

In one embodiment, the compound per se has a solubility of no more than10 μg/ml. In yet another embodiment, said solubility is no more than 1μg/ml.

In some embodiments, the compound may be a pharmaceutically activecompound, for example a cytotoxic agent. Non-limiting examples ofcytotoxic agents are those selected from calicheamycin, auristatin,doxorubicin, maytansinoid, taxol, ecteinascidin, geldanamycin and theirderivatives, and combinations thereof. Alternatively, the cytotoxicagent may be a synthetic chemotoxin made by organic synthesis and notderived from a naturally occurring compound.

In addition to the poorly soluble compound and albumin bindingpolypeptide, fusion protein or conjugate, the composition according tothis aspect of the disclosure may, in some embodiments, also comprise abinding polypeptide with an affinity for a clinically relevant target.This binding polypeptide is suitably different from the albumin bindingpolypeptide, and may be non-covalently or covalently coupled to theother components of the inventive composition. As non-limiting examples,the binding polypeptide with an affinity for a clinically relevanttarget may be selected from the group consisting of antibodies andfragments and domains thereof substantially retaining antibody bindingactivity; microbodies, maxybodies, avimers and other smalldisulfide-bonded proteins; and binding proteins derived from a scaffoldselected from the group consisting of staphylococcal protein A anddomains thereof, other three helix domains, lipocalins, ankyrin repeatdomains, cellulose binding domains, γ crystallines, green fluorescentprotein, human cytotoxic T lymphocyte-associated antigen 4, proteaseinhibitors such as Kunitz domains, PDZ domains, SH3 domains, peptideaptamers, staphylococcal nuclease, tendamistats, fibronectin type IIIdomain, transferrin, zinc fingers and conotoxins.

The composition according to the above aspect of the present disclosurehas an ability to associate with albumin in vivo or in vitro, throughthe provision in the composition of an albumin binding polypeptide, byitself or as present in a fusion protein or conjugate. In certain cases,it may be of benefit to form a complex of the composition with albuminoutside of a living organism, i.e. to add exogenous albumin to thecomposition. Such a composition may be lyophilized, providing aformulation that is suitable for storage at ambient temperature. Thus,the present disclosure also provides a composition as defined abovewhich further comprises albumin, such as human serum albumin.

The present disclosure also provides the composition according to theabove aspect for use as a medicament, i.e. for use in therapy, in caseswhere the compound is a therapeutically active compound. Suitably, theprovision of an albumin binding polypeptide, fusion protein or conjugateand optionally albumin does not deleteriously affect the therapeuticefficacy of the active compound, so the inventive composition will beuseful in those therapeutic or prophylactic settings where the compoundper se is indicated.

In another embodiment, there is provided the composition according tothe above aspect for use as a diagnostic agent, i.e. for use indiagnosis.

A related aspect of the present disclosure provides a method ofpreparation of a composition as described immediately above. The methodcomprises

providing a compound which per se has a solubility in water of no morethan 100 μg/ml; and

covalently coupling the compound to an albumin binding polypeptide,fusion protein or conjugate according aspects as described herein, thusforming a composition comprising a covalent complex of compound andalbumin binding polypeptide, fusion protein or conjugate.

In embodiments of the present disclosure where albumin is included intothe composition, the method may comprise the additional step of mixingsaid complex of compound and albumin binding polypeptide, fusion proteinor conjugate with albumin, thus forming a composition comprising anon-covalent complex of i) the covalent complex of compound and albuminbinding polypeptide, fusion protein or conjugate, and ii) albumin. Therelative proportions of the two components of this non-covalent complexmay for example be 1:1, so that one unit of the complex of poorlysoluble compound and albumin binding polypeptide, fusion protein orconjugate is associated with one molecule of albumin. In one embodiment,the method additionally comprises lyophilizing the non-covalent complexto obtain a lyophilized composition.

In another closely related aspect, the present disclosure provides amethod of increasing the aqueous solubility of a compound, comprising

providing a compound which per se has a solubility in water of no morethan 100 μg/ml;

covalently coupling the compound to an albumin binding polypeptide,fusion protein or conjugate according aspects as described herein, thusforming a covalent complex of compound and albumin binding polypeptide,fusion protein or conjugate; and

mixing said complex of compound and albumin binding polypeptide, fusionprotein or conjugate with albumin under conditions that promote thenon-covalent association of the albumin binding polypeptide withalbumin;

whereby the solubility in water of the compound in said complex isgreater than the solubility in water of the compound per se.

In these method aspects concerning the solubility of a poorly solublecompound, the optional features of the various components are asdescribed in connection with the immediately preceding compositionaspect.

While the invention has been described with reference to variousexemplary embodiments, it will be understood by those skilled in the artthat various changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor molecule to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to any particular embodiment contemplated for carryingout this invention, but that the invention will include all embodimentsfalling within the scope of the appended claims.

FIGURES

FIG. 1 is a listing of the amino acid sequences of examples of albuminbinding polypeptides of the present disclosure (SEQ ID NO:1-152, SEQ IDNO:155-203), the GA3 domain from protein G of Streptococcus strain G148extended by a N-terminal glycine residue (SEQ ID NO:153) and an albuminbinding polypeptide derived from G148-GA3 as previously described byJonsson et al (supra, SEQ ID NO:154).

FIG. 2 shows the result of binding analysis performed in a Biacoreinstrument for investigating the binding of the albumin bindingpolypeptide PEP07912 (SEQ ID NO:157) to human serum albumin. Threedifferent concentrations of purified protein (40 nM, fat gray line; 10nM, black line; and 2.5 nM, gray line) were injected over a surface with955 RU of immobilized human serum albumin.

FIGS. 3A-C show the result of binding analysis performed by ELISA forinvestigating the binding of the albumin binding polypeptides PEP07913(SEQ ID NO:153), PEP06923 (SEQ ID NO:154), PEP07271 (SEQ ID NO:155),PEP07912 (SEQ ID NO:157), PEP07554 (SEQ ID NO:156), PEP07914 (SEQ IDNO:158), PEP07968 (DOTA-conjugated PEP07911, SEQ ID NO:159) and PEP07844(SEQ ID NO:161), to IgG molecules present in 126 individual normal humansera, where A) shows the average OD-value, B) shows the percentage ofnegative sera (defined as OD<0.15), and C) shows the percentage ofpositive sera (defined as OD>1.0).

FIGS. 4A-B are chromatograms showing analysis of purified, chemicallyproduced albumin binding polypeptide PEP07834 (SEQ ID NO:160), where A)shows the absorbance signal at 220 nm, blank subtracted, and B) showsthe absorbance signal at 280 nm, blank subtracted. Two peaks appeared atboth wavelengths.

FIGS. 5A-B are spectrograms showing masspectrometric analysis of the twopeaks identified in FIGS. 4A) and B). A) is the spectrogram of the firstpeak, i.e. the monomer of PEP07834 (SEQ ID NO:160), and B) is thespectrogram of the dimer of PEP07834.

FIGS. 6A-C are diagrams showing an immunogenicity assessment of albuminbinding polypeptides PEP07913 (SEQ ID NO:153), PEP07912 (SEQ ID NO:157),PEP07914 (SEQ ID NO:158) and PEP07968 (DOTA-conjugated PEP07911, SEQ IDNO:159) in a CD3⁺ CD4⁺ T cell proliferation assay. A) shows the numberof individuals responding to the albumin binding polypeptides comparedto recombinant human albumin in a cohort of 52 Caucasian donors. B)shows the average stimulation indices (SI) for PEP07913, PEP07912,PEP07914 and PEP07968 compared to the negative control containingrecombinant human albumin. C) shows the number of responding individualsagainst all proteins in the study as compared to the buffer control.

FIGS. 7A-C shows the result of binding analysis performed in a Biacoreinstrument for investigating the binding of the albumin bindingpolypeptides A) PEP07986 (SEQ ID NO:163), B) PEP08296 (DOTA-conjugatedPEP08185, SEQ ID NO:148) and C) PEP06923 (SEQ ID NO:154) to albumin fromdifferent species. The sensorgrams shown correspond to protein injectedat a concentration of 40 nM over surfaces immobilized with albumin fromhuman (1130 RU), thin gray line; cynomolgus monkey (1046 RU), thick grayline; rat (831 RU), thick light gray line; dog (1053 RU), thin blackline; and mouse (858 RU), thick black line.

FIG. 8 shows the inhibitory effect of Z_(X)-PP013 (open circles),Z_(Y)-PP013 (open squares) and Z_(neg)-PP013 (closed triangles) oncytokine induced TF-1 cell proliferation in the presence of five timesmolar excess of HSA.

FIG. 9 shows the maximum binding responses obtained by Biacore analysisof PEP07986 (SEQ ID NO:163) stored at 4, 25 or 40° C. for one week, twoweeks, one month and three months as indicated, at a concentration of 2mg/ml, injected over immobilized HSA (704 RU) at a concentration of 10nM. Non-treated samples from time=0 are shown as references.

FIG. 10 shows the result of binding analysis performed in a Biacoreinstrument for investigating the binding of the albumin bindingpolypeptide PEP08296 (DOTA-conjugated PEP08185, SEQ ID NO:148) to humanserum albumin before and after heat treatment. Two concentrations ofPEP08296 (0.8 nM, grey lines; 4 nM, black lines) were injected over asurface with 724 RU of immobilized human serum albumin. Solid lines arebefore heat treatment and hatched lines after heat treatment for 10minutes at 90° C.

FIGS. 11A-B show the overlay of two CD spectra of PEP08296(DOTA-conjugated PEP08185, SEQ ID NO:148) before and after heattreatment for 12 min at 90° C. A) Sample incubated in PBS pH 7.2. B)Sample incubated in PBS pH 4.0.

FIG. 12 shows the maximum intensity projection (MIP) image of the wholebody distribution of ⁶⁸Ga-PEP08296 in a healthy rat, summed during 1.5 hof data collection immediately following intravenous injection (tailvein). Circulating radioactivity in the major vessels (e.g. the jugular(long arrow) and femoral (short arrow)), the heart (H), liver (L),spleen (S), kidney (K) and bladder (B) are readily delineated.

FIG. 13 shows a gel filtration chromatogram of PEP07986 (SEQ ID NO:163)injected at a concentration of 42 mg/ml, black solid line. Achromatogram of ovalbumin (Mw 43 kDa) injected at a concentration of 5mg/ml, gray broken line, is included for comparison, confirming that thepeak for PEP07986 is not an aggregate, which would have been expected inthe void volume eluted at an earlier time point than ovalbumin.

The invention will now be illustrated further through the non-limitingdescription of experiments conducted in accordance therewith. Unlessotherwise specified, conventional chemistry and molecular biologymethods were used throughout.

EXAMPLES Example 1 Cloning, Expression, Purification andCharacterization of Albumin Binding Polypeptides

In this example, ten different albumin binding polypeptides, PEP07913(SEQ ID NO:153), PEP07912 (SEQ ID NO:156), PEP07914 (SEQ ID NO:158),PEP07968 (DOTA-conjugated PEP07911, SEQ ID NO:159), PEP06923 (SEQ IDNO:154), PEP07271 (SEQ ID NO:155), PEP07554 (SEQ ID NO:156), PEP07844(SEQ ID NO:161), PEP07986 (SEQ ID NO:163) and PEP08296 (DOTA-conjugatedPEP08185, SEQ ID NO:148), the amino acid sequences of which are set outin FIG. 1 and in the appended sequence listing, were cloned, purifiedand characterized.

Material and Methods Cloning of Albumin Binding Polypeptide Variants

Mutations in G148-GA3 were generated using site directed mutagenesiswith the appropriate oligonucleotides to obtain the desired albuminbinding polypeptide variants. The gene fragments were amplified by PCRwith primers adding specific endonuclease sites as well as an N-terminalMGSS sequence preceding the albumin binding polypeptide variants. Thefragments were cleaved with NdeI and NotI, purified and ligated to acloning vector, the plasmid pAY02556 (containing an origin ofreplication from pBR322, a kanamycin resistance gene and a T7 promoterfor expression of the gene of interest), restricted with the sameenzymes. Ligations were transformed to electrocompetent E. coli TOP10cells. The transformed cells were spread on TBAB plates (30 g/l tryptoseblood agar base) supplemented with 50 μg/ml of kanamycin, followed byincubation at 37° C. overnight. The colonies were screened using PCR andsequencing of amplified fragments was performed using the biotinylatedoligonucleotide and a BigDye® Terminator v3.1 Cycle Sequencing Kit(Applied Biosystems), used in accordance with the manufacturer'sprotocol. The sequencing reactions were purified by binding to magneticstreptavidin coated beads using a Magnatrix 8000 (NorDiag AB), andanalyzed on ABI PRISM® 3100 Genetic Analyzer (PE Applied Biosystems).All albumin binding polypeptide variants were subcloned as monomers andthe constructs encoded by the expression vectors were MGSS-[PP###],where PP### corresponds to the amino acid residues constituting thesequence of the albumin binding polypeptide.

In addition, the gene fragments of G148-GA3, PP007 (SEQ ID NO:7), PP013(SEQ ID NO:13) and PP037 (SEQ ID NO:37) were amplified by PCR withprimers adding specific endonuclease sites as well as a hexahistidinsequence, a TEV protease site and a glycine residue before the aminoacid residues constituting the sequence of the albumin bindingpolypeptide. The polypeptides PEP07913 (SEQ ID NO:153), PEP07912 (SEQ IDNO:157), PEP07914 (SEQ ID NO:158) and PEP07968 (SEQ ID NO:159)correspond to the albumin binding polypeptides G148-GA3, PP007 (SEQ IDNO:7), PP013 (SEQ ID NO:13) and PP037 (SEQ ID NO:37) with glycineresidues added. The fragments were cleaved with XbaI and NotI, purifiedand ligated to a cloning vector, the plasmid pAY02512 (containing anorigin of replication from pBR322, a kanamycin resistance gene and a T7promoter for expression of the gene of interest. The cloning site ispreceded by a sequence encoding a peptide containing a hexahistidine tagfollowed by a cleavage site for the TEV protease), restricted with thesame enzymes. Ligation, transformation and sequence verification wereperformed as described above. The four albumin binding polypeptidevariants G148-GA3, PP007, PP013 and PP037 were subcloned as monomers andthe constructs encoded by the expression vectors wereMGSSHHHHHHLQSSGVDLGTENLYFQG-[PP###].

The expression vector encoding MGSSHHHHHHLQSSGVDLGTENLY-FQG-[PP013] wasfurther modified by site directed mutagenesis using oligonucleotides,resulting in the insertion of a serine residue before the amino acidresidues constituting the sequence of the albumin binding polypeptide,to obtain the construct MGSSHHHHHHLQSSGVDLGTENLYFQ-GS-[PP013]. Thisconstruct was further modified by 1) site directed mutagenesis toreplace the serine residue at position 14 (within PP013) with a cysteineresidue, generating MGSSHHHHHHLQSSGVDLGTENLYFQGS-[PP049], and 2)addition of a glycine residue C-terminally, generatingMGSSHHHHHHLQSSGVDLGTENLYFQGS-[PP049]-G. The addition of glycineC-terminally was accomplished by PCR amplification with primersincluding nucleotides encoding the glycine residue and specificendonuclease sites. The fragment was cleaved with XbaI and NotI,purified and ligated to a cloning vector, the plasmid pAY02641(containing an origin of replication from pBR322, a kanamycin resistancegene and a T7 promoter for expression of the gene of interest),restricted with the same enzymes. Ligation, transformation and sequenceverification were performed as described above.

Protein Expression

The albumin binding polypeptide variants were expressed in E. coli BL21(DE3) either with an N-terminal MGSS-extension or with an N-terminalHis₆-tag followed by a TEV-protease recognition site and a glycineresidue. A colony of each albumin binding polypeptide variant was usedto inoculate 4 ml TSB+YE medium supplemented with kanamycin to aconcentration of 50 μg/ml. The cultures were grown at 37° C. forapproximately 5 hours. 3 ml from each of the cultures was used toinoculate 800 ml TSB+YE supplemented with kanamycin to a concentrationof 50 μg/ml in parallel bio reactors (Greta system, Belach BioteknikAB). The cultivations were performed at 37° C., with aeration at 800ml/minute and an agitation profile to keep dissolved oxygen levels above30%, to an OD600 of 2, which was followed by addition of IPTG to a finalconcentration of 0.5 mM. Cultivation was continued for five hours afterwhich the cultivation was cooled to 10° C., aeration was stopped andagitation lowered to 300 rpm. Cell pellets were harvested bycentrifugation (15600×g, 4° C., 20 minutes) and stored at −20° C. untilpurification.

Purification of Albumin Binding Polypeptide Variants with a His₆-Tag anda TEV-Protease Site

Frozen cell pellets harboring soluble hexahistidine-tagged polypeptidesPEP07913 (SEQ ID NO:153), PEP07912 (SEQ ID NO:156), PEP07914 (SEQ IDNO:158), PEP07968 (SEQ ID NO:159), PEP07986 (SEQ ID NO:163) and PEP08185(SEQ ID NO:148) were suspended in 35 ml binding buffer (20 mM sodiumphosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) with an addition of 1000U Benzonase® (1.01654.001, Merck) and disrupted by ultrasonication. Foreach of the polypeptides, the ultrasonicated suspension was clarified bycentrifugation (1 h, 37000×g, 4° C.) and the supernatant was loaded ontoa His GraviTrap™ column (11-0033-99, GE Healthcare). The column waswashed with 10 ml washing buffer (20 mM sodium phosphate, 0.5 M NaCl, 60mM imidazole, pH 7.4), before eluting the polypeptide with 3 ml elutionbuffer (20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4).The buffer was exchanged to a cleavage buffer (50 mM Tris-HCl, 150 mMNaCl, pH 8) using PD-10 desalting column (17-0851-01, GE Healthcare).The amount of polypeptide product was determined by measuring theabsorbance at 280 nm before adding DTT to a final concentration of 5 mM.His₆-tagged TEV protease was added to the cleavage buffer at a 1:10 massratio relative to the polypeptide product. The cleavage was performedover night under slow mixing at 4° C. Imidazole was added to thecleavage mix, to a concentration of 20 mM, before loading the mix onto aHis GraviTrap™ column (11-0033-99, GE Healthcare) for removing cleavedHis₆-tags, His₆-tagged TEV protease and His₆-tagged uncleaved product.

For each variant, the flow-through, containing the albumin bindingpolypeptide variant, was further purified by reversed phasechromatography (RPC), as follows. The flow-through fraction was loadedon 1 ml Resource 15 RPC column (GE Healthcare), previously equilibratedwith RPC A Buffer (0.1% TFA in water). After column wash with 10 columnvolumes (CV) RPC A Buffer, bound polypeptides were eluted with a lineargradient of 0-50% RPC B Buffer (0.1% TFA in acetonitrile) during 10 CV.The flow rate was 2 ml/min and the absorbance at 280 nm was monitored.Fractions containing albumin binding polypeptide variant were identifiedby SDS-PAGE analysis and pooled.

The RPC-purified albumin binding polypeptide variants were furtherpurified by gel filtration on 120 ml Superdex 75 (GE Healthcare) packedin an XK16 column (GE Healthcare). The running buffer was 1×PBS, and theflow rate 2 ml/min. Fractions containing pure albumin bindingpolypeptide variant were pooled and concentrated to approximately 1.3mg/ml. Finally, the concentrate was purified from trace amounts ofremaining endotoxins by using 1 ml columns of AffinityPak Detoxi-GelEndotoxin removing gel (Pierce, prod#20344), according to themanufacture's recommendations.

The albumin binding polypeptide variants PEP07911 and PEP08185 wereconjugated with Mal-DOTA before the RPC-purification step, as follows.The buffer of the flow-through fraction from the IMAC-FT purificationstep was exchanged to 0.2 M NaAc, pH 5.5, using a disposable PD-10desalting column (GE Healthcare). Maleimido-mono-amide-DOTA(Macrocyclics, cat. no. B-272) was added at 5-fold molar excess andincubated for 60 minutes at 30° C. under continuous shaking. Theresulting polypeptide were denoted PEP07968 and PEP08296, respectively.

Purification of Albumin Binding Polypeptide-Variants without His₆-Tag

Frozen cell pellets harboring soluble albumin binding polypeptidevariants PEP06923 (SEQ ID NO:154), PEP07271 (SEQ ID NO:155), PEP07554(SEQ ID NO:156) and PEP07844 (SEQ ID NO:161) were suspended in 20 mMTris-HCl, pH 8 and disrupted by ultrasonication. For each of thepolypeptide variants, the ultrasonicated suspension was clarified bycentrifugation (30 min, 32000×g, 4° C.) and the supernatant was loadedonto a HSA-Sepharose column (GE Healthcare). After washing withTST-buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween 20, pH8.0), followed by 5 mM NH₄Ac, pH 5.5, bound albumin binding polypeptidevariant was eluted with 0.5 M HAc, pH 3.2.

The albumin binding polypeptide variants were further purified byreversed phase chromatography (RPC), as follows. For each of thevariants, the eluate from the HSA-affinity purification step was loadedon 1 ml Resource 15 RPC column (GE Healthcare), previously equilibratedwith RPC A Buffer (0.1% TFA in water). After column wash with 10 CV RPCA Buffer, bound polypeptides were eluted with a linear gradient of 0-50%RPC B Buffer (0.1% TFA in acetonitrile) during 10 CV. The flow rate was2 ml/min and the absorbance at 280 nm was monitored. Fractionscontaining pure albumin binding polypeptide variants were identified bySDS-PAGE analysis and pooled. Finally, the buffer was exchanged to 1×PBS(2.68 mM KCl, 137 mM NaCl, 1.47 mM KH₂PO₄, 8.1 mM Na₂HPO₄, pH 7.4) usinga disposable PD-10 desalting column (GE Healthcare).

Characterization of Purified Albumin Binding Polypeptide-Variants

The concentration was assessed by measuring the absorbance at 280 nmusing a NanoDrop® ND-1000 Spectrophotometer. The proteins were furtheranalyzed with SDS-PAGE and LC-MS.

For the SDS-PAGE analysis, approximately 10 μg of each albumin bindingpolypeptide variant was mixed with NuPAGE LDS Sample Buffer(Invitrogen), incubated at 70° C. for 15 min and loaded onto NuPAGE4-12% Bis-Tris Gels (Invitrogen). The gels were run with NuPAGE MES SDSRunning Buffer (Invitrogen) in an XCell II SureLock Electrophoresis Cell(Novex) employing the Sharp Prestained Standard (Invitrogen) asmolecular weight marker and using PhastGel BlueR (GE Healthcare) forstaining.

To verify the identity of the albumin binding polypeptide variants,LC/MS analyses were performed using an Agilent 1100 LC/MSD system,equipped with API-ESI and a single quadruple mass analyzer.Approximately 10 μg of each of the purified albumin binding polypeptidevariants was loaded on a Zorbax 300SB-C8 Narrow-Bore column (2.1×150 mm,3.5 μm, Agilent Technologies) at a flow-rate of 0.5 ml/min. Polypeptideswere eluted using a linear gradient of 10-70% solution B for 15 min at0.5 ml/min. The separation was performed at 30° C. The ion signal andthe absorbance at 280 and 220 nm were monitored. The molecular weightsof the purified albumin binding polypeptide variants were confirmed byMS.

Results

The expression levels of the albumin binding polypeptide variants were10-30 mg product/g cell pellet, as estimated from SDS-PAGE analysis.

For all variants, the purity, as determined by SDS-PAGE analysis,exceeded 95% and the LC/MS analysis verified the correct molecularweights. After purification, between 1 and 8 mg of pure polypeptide wasobtained for each of the ten albumin binding polypeptide variants.

Example 2 Affinity Determination for Albumin Binding Polypeptides

In this example, PEP06923 (SEQ ID NO:154), PEP07271 (SEQ ID NO:155),PEP07844 (SEQ ID NO:161), PEP07912 (SEQ ID NO:157), PEP07913 (SEQ IDNO:153), PEP07914 (SEQ ID NO:158) and PEP07968, (DOTA-conjugatedPEP07911, SEQ ID NO:159), synthesized or expressed and purified inExample 1 were characterized for affinity to human serum albumin (HSA)using a Biacore instrument. PEP07913 corresponds to the amino acidsequence of G148-GA3 with addition of a N-terminal glycine residue,whereas PEP07271, PEP07844, PEP07912, PEP07914 and PEP07968 correspondto the albumin binding polypeptides of PP001 (SEQ ID NO:1), PP043 (SEQID NO:43), PP007 (SEQ ID NO:7), PP013 (SEQ ID NO:13) and PP037 (SEQ IDNO:37) with different N-terminal amino acid additions.

Material and Methods

Biosensor analysis on a Biacore2000 instrument (GE Healthcare) wasperformed with HSA (Albucult®, Novozymes), immobilized by amine couplingonto the carboxylated dextran layer of the surfaces of CM-5 chips(research grade; GE Healthcare) according to the manufacturer'srecommendations. Surface 1 of the chip was activated and deactivated andused as a reference cell (blank surface) during injections, whereassurface 2 comprised HSA immobilized to 731 resonance units (RU) andsurface 4 comprised HSA immobilized to 955 RU. The purified albuminbinding polypeptide variants were diluted in running buffer HBS-EP (GEHealthcare) to 2.5 nM, 10 nM and 40 nM, and injected at a constantflow-rate of 50 μl/min for 5 minutes, followed by injection of HBS-EPfor 60 minutes. The surfaces were regenerated with one injection of 25μl HCl, 10 mM. The affinity measurements were performed in two sets; inthe first set HBS-EP, PEP06923, PEP07271, PEP07912, PEP07913, PEP07914and PEP07968 were injected (chip surface 2), and in the second setHBS-EP, PEP06923, PEP07844, PEP07912 and PEP07914 were injected (chipsurface 4). PEP06923 was injected twice in each run as a control. Theresults were analyzed with a BiaEvaluation software (GE Healthcare).Curves of the blank surface were subtracted from the curves of theligand surfaces.

Results

The Biacore 2000 instrument has a technical limitation, hinderingmeasurements of very high affinity. Hence, the purpose of the Biacorestudy was not to determine the exact kinetic parameters of the albuminbinding polypeptide variants' affinity for HSA. However, the resultsprovide a quantitative estimation of the relative affinities of thesepolypeptides for albumin. After subtraction of reference surface andbuffer injection, curves were fitted to a 1:1 (Langmuir) binding modelusing BIAevaluation software with correction for mass transfer and withRUmax set as a local parameter. Curves are shown in FIG. 2. The relativeK_(D), k_(a) (k_(on)) and k_(d) (k_(off)) values were estimated and arepresented in the Tables below.

TABLE 1 Kinetic parameters (k_(a), k_(d) and K_(D)) of albumin bindingpolypeptides to HSA, 1st set k_(a) (Ms⁻¹) k_(d) (s⁻¹) K_(D) (M) PEP079135.7 × 10⁵ 9.3 × 10⁻⁴ 1.6 × 10⁻⁹  PEP06923 (1) 2.9 × 10⁷ 2.9 × 10⁻⁵ 9.9 ×10⁻¹³ PEP06923 (2) 2.6 × 10⁷ 2.8 × 10⁻⁵ 1.1 × 10⁻¹² PEP07271 3.9 × 10⁶2.9 × 10⁻⁵ 7.5 × 10⁻¹² PEP07912 4.6 × 10⁶ 2.8 × 10⁻⁵ 6.2 × 10⁻¹²PEP07914 3.5 × 10⁶ 2.5 × 10⁻⁵ 7.2 × 10⁻¹² PEP07968 3.0 × 10⁶ 2.7 × 10⁻⁵9.0 × 10⁻¹²

TABLE 2 Kinetic parameters (k_(a), k_(d) and K_(D)) of albumin bindingpolypeptides to HSA, 2nd set k_(a) (Ms⁻¹) k_(d) (s⁻¹) K_(D) (M) PEP06923(1) 2.0 × 10⁷ 2.6 × 10⁻⁵ 1.3 × 10⁻¹² PEP06923 (2) 2.1 × 10⁷ 2.5 × 10⁻⁵1.2 × 10⁻¹² PEP07912 5.4 × 10⁶ 2.8 × 10⁻⁵ 5.2 × 10⁻¹² PEP07914 3.8 × 10⁶2.6 × 10⁻⁵ 6.9 × 10⁻¹² PEP07844 5.4 × 10⁶ 2.3 × 10⁻⁵ 4.4 × 10⁻¹²

As shown in Tables 1 and 2, PEP07271 (SEQ ID NO:155), PEP07844 (SEQ IDNO:161), PEP07912 (SEQ ID NO:157), PEP07914 (SEQ ID NO:158) and PEP07968(DOTA-conjugated PEP07911, SEQ ID NO:159) all seem to have approximatelythe same affinity for HSA, widely exceeding the affinity of the parentG148-GA3 (PEP07913; SEQ ID NO:153). The HSA affinity of thesepolypeptides is slightly lower compared to PEP06923 (SEQ ID NO:154),despite similar off-rate.

Example 3 Determination of Melting Temperature (Tm) for Albumin BindingPolypeptides

In this example, the albumin binding polypeptide variants PEP07913 (SEQID NO:153), PEP06923 (SEQ ID NO:154), PEP07271 (SEQ ID NO:155), PEP07554(SEQ ID NO:156), PEP07912 (SEQ ID NO:157), PEP07914 (SEQ ID NO:158),PEP07968 (DOTA-conjugated PEP07911, SEQ ID NO:159), PEP07844 (SEQ IDNO:161) and PEP07986 (SEQ ID NO:163), expressed and purified asdescribed in Example 1, and the albumin polypeptide variant PEP07975(DOTA-conjugated PEP07834, SEQ ID NO:160), produced as described inExample 5, were analyzed by CD analysis. PEP07913 corresponds to thesequence of G148-GA3 having an N-terminal glycine residue, PEP06923 isan engineered high affinity derivative previously described by Jonssonet al, supra, whereas PEP07271, PEP07554, PEP07912, PEP07914, PEP07968,PEP07844 and PEP07975 are examples of the albumin binding polypeptidesof PP001 (SEQ ID NO:1), PP007 (SEQ ID NO:7), PP013 (SEQ ID NO:13), PP037(SEQ ID NO:37) and PP043 (SEQ ID NO:43) having different N-terminalamino acid additions according to the present disclosure.

Material and Methods

Purified albumin binding polypeptide variants were diluted in 1×PBS, tofinal concentrations between 0.4 and 0.5 mg/ml. Circular dichroism (CD)analysis was performed on a Jasco J-810 spectropolarimeter in a cellwith an optical path-length of 1 mm. In the variable temperaturemeasurements, the absorbance was measured at 221 nm from 20° C. to 90°C., with a temperature slope of 5° C./min.

Results

The melting temperatures (Tm) of the different albumin bindingpolypeptide variants were calculated by determining the midpoint of thetransition in the CD vs. temperature plot. The results are summarized inTable 3 below.

TABLE 3 Determined Tm values of tested albumin binding polypeptidevariants N-terminal Variant SEQ ID NO: # sequence³ Tm (° C.) PEP07913SEQ ID NO: 153 GL 61 PEP06923 SEQ ID NO: 154 GSSL 57 PEP07271 SEQ ID NO:155 GSSL 65 PEP07554 SEQ ID NO: 156 GSSL 58 PEP07912 SEQ ID NO: 157 GL53 PEP07914 SEQ ID NO: 158 GL 59 PEP07968 SEQ ID NO: 159¹ GL 53 PEP07975SEQ ID NO: 160^(1, 2) AL 50 PEP07844 SEQ ID NO: 161 GSSL 65 PEP07986 SEQID NO: 163 GSL 61 ¹The peptide is conjugated with maleimide-DOTA at thecysteine ² The peptide is amidated at the C-terminus ³Leucine(underlined) is the residue in position 1 of the amino acid sequence ofthe albumin binding polypeptide as defined in the first aspect of thepresent disclosure

The polypeptide PEP07968 is identical to PEP07912, except for the formerhaving a cysteine residue in position 14 conjugated with maleimide DOTA,and the latter a serine residue. Thus, the DOTA modification should notaffect the melting temperature. Also PEP07975 is conjugated with DOTAusing C₁₄, and is identical to PEP07968 except for the C-terminal amide(resulting from the peptide synthesis in Example 5) and for having anN-terminal alanine instead of a glycine. Furthermore, comparing PEP07912and PEP07554 reveals that an N-terminal serine gives a higher meltingtemperature than a glycine in the same position (5° C. difference inTm). Thus, all albumin binding polypeptide variants according to thepresent disclosure show Tm above 55° C., except PEP07912 andDOTA-conjugated variants. Taking into consideration the importance ofthe N-terminal portion, all the tested albumin binding polypeptides aresuperior to the prior art derivative of Jonsson et al, i.e. PEP06923.

Example 4 Serum Response Analysis

The percentage of human serum containing IgG, capable of binding to aset of albumin binding polypeptides as disclosed herein was analyzed byELISA. In total, 149 serum samples corresponding to 127 individuals werescreened.

Material and Methods

ELISA plates (96-well, half area plates (Costar, cat. No. 3690)) werecoated with 50 μl/well of Albucult® (Novozymes) diluted to 8 μg/ml incoating buffer (Sigma, cat. No. 3041). The plates were coated over nightfor three days at 4° C. On the day of analysis, the plates were washedtwice with tap water and blocked for 2 hours with 100 μl of phosphatebuffered saline (PBS) containing 0.05% casein (PBSC). The plates wereemptied and 50 μl/well of the albumin binding polypeptides PEP07913 (SEQID NO:153), PEP06923 (SEQ ID NO:154), PEP07271 (SEQ ID NO:155), PEP07912(SEQ ID NO:157), PEP07554 (SEQ ID NO:156), PEP07914 (SEQ ID NO:158),PEP07968 (DOTA-conjugated PEP07911, SEQ ID NO:159) and PEP07844 (SEQ IDNO:161), diluted to 2 μg/ml in PBSC were added according to a pre-madeplate layout. After incubation for two hours at room temperature (RT),the plates were washed in PBSC four times using an automated ELISAwasher. The 149 serum samples from 129 individuals were diluted 50 timesin PBSC by adding 24 μl serum to 1174 μl PBSC. 50 μl of the diluted serawas added per well according to the pre-made plate layout. Each serumsample was tested as a singlet. Positive and negative controls wereincluded on each plate and for each albumin binding polypeptide. Albuminbinding antibodies (50 μl, 0.5 μl/ml immunoglobulin solution prepared inhouse from sera from primates immunized with PEP06923) was added as apositive control and 50 μl PBSC was used as a negative control. Theplates were incubated for one hour at RT and subsequently washed fourtimes in PBSC using an automated ELISA washer. The bound IgG wasdetected with 50 μl/well of anti-human IgG (Southern Biotech, cat no2040-05) diluted 10 000 times in PBSC. After washing four times in PBSCusing an automated ELISA washer, 50 μl/well of substrate was added(Pierce cat. No. 34021). The reaction was stopped after 10-15 minutes bythe addition of 50 μl H₂SO₄ to each well, prior to measuring theabsorbance using a multi-well plate reader (Victor3, Perkin Elmer).

Results

Of the 149 sera screened for IgG binding to the albumin bindingpolypeptides, 23 were negative for all eight polypeptides(OD-value<0.1), i.e. showed no IgG bound to the polypeptides. Theanalysis was performed with the 126 sera that were positive for one ormore albumin binding polypeptides. The average absorbance was calculated(FIG. 3A) and the percentage of sera with OD-values values either <0.15(FIG. 3B) or >1.0 (FIG. 3C). The highest average OD-value and thehighest percentage of serum with IgG binding were obtained with PEP07913(SEQ ID NO:153), PEP06923 (SEQ ID NO:154) and PEP07844 (SEQ ID NO:161),whereas least reactivity was found against PEP07968 (DOTA-conjugatedPEP07911, SEQ ID NO:159), PEP07914 (SEQ ID NO:158) and PEP07954 (SEQ IDNO:156).

Thus, the most reactive albumin binding polypeptides were the parentalG148-GA3 (PEP07913, SEQ ID NO:153) and the previously affinity improvedderivative (PEP06923, SEQ ID NO:154), having helix 1 retained fromG148-GA3. The third of the more reactive polypeptides (PEP07844, SEQ IDNO:161) contains the original lysine in position 14 in helix 1. Thisresidue is intended for conjugation, and will therefore not be exposedin the final context. The identical albumin binding polypeptide variant,except for having an alanine in position 14 (PEP07554, SEQ ID NO:156),is one of the least reactive.

Example 5 Chemical Synthesis of a DOTA-Conjugated Albumin BindingPolypeptide Material and Methods

The albumin binding polypeptide PEP07834 (SEQ ID NO:160) was synthesizedby solid phase peptide synthesis (SPPS, as described by Quibell, M. &Johnson, T., in Fmoc Solid Phase Peptide Synthesis—A Practical Approach,W. C. Chan, P. D. White Eds, Oxford University Press 2000, 115-135) in a433 A Peptide Synthesizer reactor (Applied Biosystems, Foster City,Calif.) on a 0.1 mmol scale, i.e. with a theoretical possible yield of0.1 mmol peptide, using standard Fmoc chemistry. An acid-labile Fmocamide resin was used as solid support throughout the synthesis (RinkAmide MBHA Resin LL (100-200 mesh), loading 0.39 mmol amide/g resin(Novabiochem)).

47 amino acid residues according to the sequence below were coupled tothe amide resin by acylation reactions in the reactor for 10 minutes atroom temperature (RT) and mixing. The acylation reactions were performedwith a ten-fold molecular excess of Fmoc protected amino acids in NMP(N-methylpyrrolidone, Merck), activated with 1 eq of2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate(HBTU, IRIS Biotech), 1 eq of 1-hydroxybenzotriazole (HOBt, IRISBiotech) and 2 eq of diisopropylethylamine (DIEA, Applied Biosystems).In addition, all reactive amino acid side chains were protected withstandard side chain protection groups (tert-butyl (tBu) for Asp, Glu,Ser, Thr and Tyr, tert-butyloxycarbonyl (Boc) for Lys,2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for Arg, andtrityl (Trt) for Asn and Cys) prior to activation and coupling. In orderto diminish the amount of incomplete couplings leading to truncatedpeptides, a minor amount of selected amino acid residues were subjectedto coupling by acylation twice, without Fmoc deprotection as describedbelow between the first and second coupling. The amino acid sequence ofthe synthesized albumin binding polypeptide PEP07834 was

(SEQ ID NO: 160-NH₂) ALASAKEAAN AELDCYGVSD FYKRLIDKAK TVEGVEALKD AILAALP-NH₂.The underlined amino acid residues were double coupled. Any remainingunreacted amino groups on the resin bound peptides were capped withacetic anhydride (0.5 M acetic anhydride (AlfaAesar), 0.125 M DIEA,0.015 M HOBt in NMP) for 5 min. Following every coupling, deprotectionof the N-terminal Fmoc group on the resin bound peptides were performedby treatment with 20% piperidine (Sigma-Aldrich) in NMP for 10 min.

After completed synthesis, the peptides were cleaved from the solidsupport and simultaneously the side chain protection groups were cleavedoff by treatment with TFA/EDT/H₂O/TIS (94:2.5:2.5:1) (TFA:trifluoroacetic acid (Apollo), EDT: 1,2-ethanedithiol (Aldrich), TIS:triisopropylsilane (Aldrich)) at RT for 2 h with occasional mixing.After TFA treatment, the peptides were extracted three times using 20%acetonitrile (Merck) in water and tert-butyl methyl ether (Merck). Theaqueous phases were combined, filtered and lyophilized.

The crude peptides were analyzed and purified by semi-preparativeRP-HPLC (Reprosil GOLD C18 300, 250*10 mm, 5 μm particle size) and agradient of 32-55% B (A: 0.1% TFA-H₂O; B: 0.1% TFA-CH₃CN) during 25 minat a flow rate of 2.5 ml min⁻¹, followed by lyophilization.

The synthetic yield was determined by calculation of the integratedareas under the peaks from the 220 nm signal from the crude analysis onRP-HPLC. The correct molecular weight was verified using liquidchromatography electrospray ionization mass spectrometry (LC-ESI-MS) ona 6520 Accurate Mass Q-TOF LC/MS (Agilent Technologies). The purity ofthe product was verified using RP-HPLC (Reprosil GOLD C18 300, 250*4.6mm, 3 μm particle size) using a gradient of 35-55% B over 25 min at aflow rate of 1.0 ml min⁻¹.

DOTA Conjugation

3 mg of PEP07834-amide (SEQ ID NO:160-amide) was reduced with 20 mM DTTat 40° C. for 30 minutes. Excess DTT was removed by buffer exchange on aPD-10 column (GE Healthcare) to 0.2 M ammonium acetate, pH 5.5. Thecoupling was performed with a 5-fold molar excess of chelator,maleimido-mono-amide-DOTA (Macrocyclics, Cat. No. B-272) solution inwater (1 mg/ml). The mixture was incubated for 1 hour at 30° C. undercontinuous shaking. Purification from non-conjugated chelators was madeon a semi-preparative RPC column (Zorbax 300SB C18, 9.4×250 mm, 5 μm).The coupling degree of the purified material was analyzed by HPLC-MS ona Zorbax 300SB C8 150×2.1 mm, 3.5 μm analytical column. Onlymaleimide-DOTA-conjugated PEP07834, denoted PEP07975, was detected bythe method.

Results

Based on the elution profile of the crude material, the synthetic yieldof the albumin binding polypeptide PEP07834-amide (SEQ ID NO:160-amide)was determined to be 8%. The found molecular weight was 4952.9 Da, whichis in good agreement with the theoretical molecular weight calculated to4952.6 Da. When analyzing the purified product, approximately 10-15% ofthe protein was found to be a disulfide linked homodimer (FIGS. 4 and5). The binding activity of the DOTA-conjugated peptide (PEP07975) wasconfirmed as described in Example 2 (data not shown), and the meltingtemperature determined as described in Example 3.

Example 6 Immunogenicity Testing of Albumin Binding Polypeptides

PEP07913 (SEQ ID NO:153), PEP07912 (SEQ ID NO:157), PEP07914 (SEQ IDNO:158), and PEP07968 (DOTA-conjugated PEP07911, SEQ ID NO:159) werescreened for their ability to induce T cell proliferation in peripheralblood mononuclear cells (PBMC) from 52 human Caucasian individuals(obtained from CRI-Labo Medische Analyse, Gent, Belgium). PEP07913corresponds to the sequence of G148-GA3 having an N-terminal glycineresidue, whereas PEP07912, PEP07914 and PEP07968 are examples of thealbumin binding polypeptides of PP007 (SEQ ID NO:7), PP013 (SEQ IDNO:13) and PP037 (SEQ ID NO:37) having different N-terminal amino acidadditions according to the present disclosure.

Materials and Methods

PBMCs, prepared according to standard cell biological methods, wereadded to a tissue culture (TC) treated 96-well round bottom plate(Falcon) in an amount of 300 000 PBMCs/well. The cells were stimulatedby addition of 100 μl/well of albumin binding polypeptides PEP07913,PEP07912, PEP07914 and PEP07968 in AIMV medium (Invitrogen) additionallycontaining 900 μg/ml (3-fold molar excess) of recombinant human albumin(Albucult®, Novozymes). This corresponded to a final concentration ofalbumin binding polypeptide of 30 μg/ml. The stimulation was done ineight-plicates, i.e. the same albumin binding polypeptide were added toeight wells in identical amounts and under the same conditions. Inpositive control wells, the cells were stimulated with either 30 μg/mlKeyhole Limpet Hemocyanin (KLH, Calbiochem) or 30 μg/ml tetanus toxoid(TT, Statens Serum Institut). In negative control wells, only AIMVmedium with or without 900 μg/ml of albumin were added.

Cell proliferation was assessed after seven days of culturing usingAlexa Fluor 488 Click-iT EdU flow cytometry assay kit (Invitrogen). 1μM/well of EdU incorporation marker was added on day six. On day seven,cells were washed, dissociated from the plate, washed again and stainedfor 30 minutes with anti-CD3-PerCP reagent (Becton Dickinson) andanti-CD4-Alexa647 reagent (Becton Dickinson). Following staining, thecells were washed, fixed (BD cellfix, BD biosciences), permeabilized(using saponin) and stained for EdU by addition of Click-iT reagentaccording to the manufacturer's protocol (Invitrogen). After completedstaining, cells were washed again and analyzed using flow cytometry(FACSCantoll, BD Biosciences). To assess the number of proliferatingcells, a fixed number of fluospheres (Invitrogen) was added to each wellbefore analysis. All staining procedures and washes were performeddirectly in the 96-well plate.

The raw FACSCantoll data were gated hierarchically on CD3⁺ CD4⁺ T cellsand the number of gated cells as well as their fluorescence intensity ofEdU-Alexa Flour 488 incorporation marker were recorded. The mean valuesof the number of proliferating cells/eight-plicate of protein treatedwells were compared to the positive and negative controls and theresulting ratios, described as stimulation indices (SI), werecalculated. Based on the SI and the variation between replicates,threshold SI-values were set to 2.0 and 0.5 for stimulation andinhibition, respectively.

Results

The albumin binding polypeptides PEP07913, PEP07912, PEP07914 andPEP07968 were assessed for their immunogenic potential in the presenceof 3-fold excess of recombinant human albumin in a target humanpopulation using an in vitro PBMC proliferation assay. Compared to thealbumin control, PEP07913 induced CD3⁺CD4⁺ T cells proliferation in 6 of52 donors, PEP07912 in 5 of 52 donors and PEP07914 and PEP07968 in 1 of52 donors (FIG. 6A).

The mean stimulation index (SI) for all 52 donors was not significantlydifferent for PEP07914 and PEP07968 compared to the negative controlcontaining recombinant human albumin (p=0.79 and 0.48 respectively, FIG.6B). The SI for PEP07913 was significantly higher (p=0.002) whereas theSI for PEP07912 was higher but not significant (p=0.03, FIG. 6B).

As compared to buffer only, the number of responding individuals was 10for PEP07912, 7 for PEP07912, 2 for PEP07914, 1 for PEP07968, 2 forrecombinant human albumin, and 49 and 51 for the two positive controlsTT and KLH, respectively (FIG. 6C). The albumin binding polypeptideswere ranked according to their immunogenicity in the following order:PEP07913>PEP07912>PEP07914>PEP07968. Both PEP07914 and PEP07968 weredefined as non-immunogenic. The above results thus demonstrate that theimmunogenic potential of the albumin binding polypeptides of the presentdisclosure is low, as compared to the positive controls.

Example 7 Albumin Binding Polypeptides' Affinity to Albumin fromDifferent Species

In this example, PEP06923 (SEQ ID NO:154), PEP07986 (SEQ ID NO:163) andPEP08296, (DOTA-conjugated PEP08185, SEQ ID NO:148), expressed andpurified as described in Example 1, were characterized for affinity toalbumin from human (HSA), cynomolgus monkey (CSA), rat (RSA), mouse(MSA) and dog (DSA) using a Biacore instrument.

Material and Methods

Biosensor analysis on a Biacore2000 instrument (GE Healthcare) wasperformed with HSA (Albucult®, Novozymes), CSA (purified in-house fromcynomolgus serum), RSA (Sigma-Aldrich, Cat. No. A6272), MSA(Sigma-Aldrich, Cat. No. A3559) and DSA (MP Biomedicals, Cat. No.55925), immobilized by amine coupling onto the carboxylated dextranlayer of the surfaces of CM-5 chips (research grade; GE Healthcare)according to the manufacturer's recommendations.

On chip 1, surface 1 was activated and deactivated and used as areference cell (blank surface) during injections, whereas surface 2comprised HSA immobilized to 1130 resonance units (RU), surface 3comprised CSA immobilized to 1046 RU, surface 4 comprised RSAimmobilized to 831 RU. On chip 2, surface 1 was used as blank surface,whereas surface 3 comprised MSA immobilized to 858 RU. On chip 3,surface 1 was used as blank surface, whereas surface 2 comprised DSAimmobilized to 1053 RU.

For analysis of affinity for HSA, CSA, and RSA (chip 1), the purifiedalbumin binding polypeptide variants were diluted in running bufferHBS-EP (GE Healthcare) to 40 nM, 10 nM and 2.5 nM; for analysis ofaffinity for MSA (chip 2) the albumin binding polypeptide variants werediluted to 1280 nM, 640 nM, 160 nM and 40 nM and for analysis ofaffinity for DSA (chip 3) albumin binding polypeptide variants werediluted to 1280 nM, 640 nM, 160 nM, 40 nM and 10 nM. The albumin bindingpolypeptides were injected at a constant flow-rate of 50 μl/min for 5minutes, followed by injection of HBS-EP for 60 minutes. The surfaceswere regenerated with one injection of 25 μl HCl, 10 mM. All sampleswere run in duplicates.

The results were analyzed with a BIAevaluation software (GE Healthcare).Curves of the blank surface were subtracted from the curves of theligand surfaces.

Results

The Biacore 2000 instrument has a technical limitation, hinderingmeasurements of very high affinity. Hence, the purpose of the Biacorestudy was not to determine the exact kinetic parameters of the albuminbinding polypeptide variants' affinity for HSA, CSA, RSA, MSA and DSArespectively. However, the results provide a quantitative estimation ofthe relative affinities of the enclosed polypeptides for albumin fromthese different species. After subtraction of reference surface andbuffer injection, curves were fitted to a 1:1 (Langmuir) binding modelusing BIAevaluation software with correction for mass transfer and withRUmax set as a local parameter. Representative binding curves are shownin FIG. 7.

PEP07986 and PEP08296 (DOTA-conjugated PEP08185) bind with high affinity(K_(D) in the range from below picomolar to below nanomolar) to humanserum albumin as well as to albumin from the frequent preclinical modelspecies rat, cynomolgus monkey, mouse and dog. The relative affinitiesfor the different species can be ranked as RSA HSA/CSA>MSA/DSA, i.e. theK_(D) values ranked asK_(D-RSA)≦K_(D-HSA)/K_(D-CSA)<K_(D-MSA)/K_(D-DSA). The affinities interms of K_(D) values are the same or slightly lower (but in the sameorder of magnitude) as the affinity obtained for PEP06923 (non-inventivepolypeptide).

Example 8 In Vitro Activity of Protein Z Variants Fused to an AlbuminBinding Polypeptide

In this example, polypeptides comprising cytokine-specific protein Z(derivative of domain B of staphylococcal protein A) variantsgenetically fused to the albumin binding polypeptide variant PP013 (SEQID NO:13) were tested for their functionality, this being to blockcytokine-induced proliferation of TF-1 cells in the presence of humanserum albumin. Proliferation of TF-1 cells is dependent of the presenceof any of several different types of cytokines and the proliferativeresponse can be inhibited by blocking reagents such as the correspondingcytokine-specific protein Z variant. PP013 fused to a protein Z variantwith specificity for an irrelevant protein was used as negative control.

Materials and Methods Cloning of Z-PP013 Fusion Proteins

Gene fragments of protein Z variants with specificity for cytokine X orY respectively, or for an irrelevant protein (negative control), wereamplified by PCR using primers adding PstI and Accl specificendonuclease sites. The fragments were cleaved with PstI and Accl,purified and ligated into an expression vector, the plasmid pAY02747,restricted with the same enzymes. pAY02747 contains an origin ofreplication from pBR322, a kanamycin resistance gene and a T7 promoterfor expression of the gene of interest. The cloning site is preceded bya sequence encoding the amino acids MGSSLQ and succeeded by a sequenceencoding VDSS-PP013, where PP013 is the disclosed albumin bindingpolypeptide with SEQ ID NO:13. Ligation, transformation and sequenceverification were performed as described above. The encoded proteinswere:

1) MGSSLQ-Z_(X)-VDSS-PP013 (denoted Z_(X)-PP013)

2) MGSSLQ-Z_(Y)-VDSS-PP013 (denoted Z_(Y)-PP013)

3) MGSSLQ-Z_(neg)-VDSS-PP013 (denoted Z_(neg)-PP013)

Protein Expression

Z_(X)-PP013, Z_(Y)-PP013 and Z_(neg)-PP013 were expressed in E. coliBL21 (DE3) cells. Colonies from the transformations of each fusionvariant were used to inoculate starter cultures of 50 ml TSB+YE mediumsupplemented with kanamycin to a concentration of 50 μg/ml. The cultureswere grown at 37° C. over night with agitation, 100 rpm. The startercultures were then used to inoculate 900 ml TSB+YE medium supplementedwith kanamycin to a concentration of 50 μg/ml. The cultures were grownfor approximately 1.5 h to an OD600 of >1.1, upon which IPTG was addedto a final concentration of 0.2 mM. Cultivation was continued for fivehours. Cell pellets were harvested by centrifugation (15600 g, 4° C., 20minutes) and stored at −20° C. until purification.

Protein Purification

Frozen cell pellets harboring soluble fusion protein variantsZ_(X)-PP013, Z_(Y)-PP013 and Z_(neg)-PP013 were resuspended in 50 mMTris-HCl, 150 mM NaCl, pH 8 and 1000 U Benzonase® (Merck Cat. No.1.01654.0001) was added. The cells were disrupted by ultrasonication andfor each of the fusion protein variants, the ultrasonicated suspensionwas clarified by centrifugation (15 min, 37000 g, 4° C.). 20×TST-buffer(20×[25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween 20, pH 8.0])was added at a volume resulting in 1×TST buffer in the clarifiedsuspension. Each sample of fusion protein variant was loaded onto aHSA-Sepharose column (GE Healthcare). After washing with TST-buffer,followed by 5 mM NH₄Ac, pH 5.5, bound fusion protein variant was elutedwith 0.5 M HAc, pH 2.5.

The fusion protein variants were further purified by reversed phasechromatography (RPC), as follows. For each of the variants, the eluatefrom the HSA-affinity purification step was loaded on a 1 ml Resource 15RPC column (GE Healthcare) previously equilibrated with RPC A Buffer(0.1% TFA in water). After column wash with 10 CV RPC A Buffer and 5 CVof RPC B Buffer (0.1% TFA in acetonitrile), bound fusion proteins wereeluted with a linear gradient of 10-50% RPC B Buffer over 20 CV. Theflow rate was 2 ml/min and the absorbance at 280 nm was monitored.Fractions containing pure fusion protein variants were identified bySDS-PAGE analysis and pooled. Finally, the buffer was exchanged to 1×PBS(2.68 mM KCl, 137 mM NaCl, 1.47 mM KH₂PO₄, 8.1 mM Na₂HPO₄, pH 7.4) usinga disposable PD-10 desalting column (GE Healthcare). To verify theidentity of the fusion protein variants, SDS-PAGE and LC/MS analyseswere performed as described in Example 1.

In Vitro Cell Assay of Z-PP013 Fusion Proteins

The cell line TF-1 (CLS Cat. No. 300434) was propagated as recommendedby the provider in RPMI 1640 medium+10% fetal calf serum (Gibco) withthe addition of 2 ng/ml of rhGM-CSF (Miltenyi). At the day ofexperiment, the cells were washed in RPMI 1640 medium+10% fetal calfserum to remove GM-CSF.

The ability of Z_(X)-PP013 and Z_(Y)-PP013 to block cytokine inducedproliferation was analyzed by mixing the molecules Z_(Y)-PP013,Z_(Y)-PP013 and Z_(neg)-PP013 with cytokines X and Y respectively, andwith a five times molar excess of HSA (Albucult®, Novozymes). Themolecules were titrated in a 2-fold dilution series with a fixedconcentration of cytokine (4.9 pM) and a five times molar excess of HSA.The titration was performed in 96-well plates in a volume of 100 μl. 25000 cells were added per well (100 μl) and plates were incubated at 37°C., 5% CO₂ for three days. To measure the proliferation, 19 μl of CCK-8cell proliferation reagent (Sigma) diluted two times in RPMI 1640medium+10% fetal calf serum, was added per well. The color reaction wasmonitored after 4 hours using 96-well plate reader (Victor3;PerkinElmer).

Results

As shown in FIG. 8, both Z_(X)-PP013 and Z_(Y)-PP013 inhibited therespective cytokine induced proliferation in the presence of HSA whereasZ_(neg)-PP013, the negative control, did not affect proliferation ofTF-1. Thus, the experiment shows that the function of the Z moleculeswas retained when incorporated into a fusion protein containing thealbumin binding polypeptide, and also when the fusion proteins werebound to albumin.

Example 9 Long-Term Stability of an Albumin Binding Polypeptide

In this example, the stability of PEP07986 (SEQ ID NO:163), expressedand purified as described in Example 1, was investigated after storageat 4, 25, and 40° C. for up to three months. The status of thepolypeptide after storage was investigated by measuring its binding toHSA using a Biacore instrument.

Material and Methods

Lyophilized PEP07986 was dissolved in sterile NaPi buffer (20 mM sodiumphosphate, 150 mM sodium chloride, pH 7.2) at a concentration of 2mg/ml. A reference sample (time=0) was removed and stored at −80° C.Aliquots of 105 μl were stored in sterile screw-cap eppendorf tubessealed with parafilm at 4, 25, and 40° C. After one week, two weeks, onemonth and three months, a sample stored at each temperature was cooledto 4° C., centrifuged for 5 min at 13000 rpm and then stored at −80° C.awaiting Biosensor analysis.

Biosensor analysis was performed essentially as described in Example 2but with HSA (Albucult®, Novozymes), immobilized to 704 resonance units(RU) and the albumin binding polypeptide variant was diluted to 10 nMand injected at a constant flow-rate of 20 μl/min for 10 minutes,followed by injection of HBS-EP for 10 minutes.

Results

The binding to HSA of PEP07986 (SEQ ID NO:163) was retained afterstorage at 4, 25, and 40° C. for at least three months. The maximumbinding responses to HSA obtained for PEP07986 stored at the variousconditions are shown in FIG. 9.

Example 10 Stability of an Albumin Binding Polypeptide Under ExtremeConditions

In this example, biosensor and circular dichroism (CD) analysis of thealbumin binding polypeptide PEP08296 (DOTA-conjugated PEP08185, SEQ IDNO:148) after heat treatment (90° C.) in low pH (˜4.0) buffer isdescribed. Since such extreme reaction conditions have to be used forexample for ⁶⁸Ga labeling of DOTA-modified proteins, the influence ofhigh heat and low pH treatment on the structural identity of thepolypeptide and its capacity to bind HSA was investigated by measuringthe melting temperature (Tm), refolding properties and binding to HSA.

Material and Methods Biosensor Analysis of Heat Stability

Biosensor analysis on a Biacore 2000 instrument (GE Healthcare) wasperformed with HSA (Albucult®, Novozymes) immobilized by amine couplingonto the carboxylated dextran layer of the surfaces of CM-5 chip(research grade; GE Healthcare) according to the manufacturer'srecommendations. Surface 1 of the chip was activated and deactivated andused as a reference cell (blank surface) during injections, whereassurface 2 comprised HSA immobilized to 724 resonance units (RU).PEP08296 (50 μl, 100 μg) in a 15 ml Falcon tube was diluted with 450 μl0.2 M sodium acetate (NaAc) pH 5.5 to a final peptide concentration of0.2 mg/mL. After addition of 1.5 ml 0.05 M HCl (resembling theconditions and volume used for eluting a ⁶⁸Ge/⁶⁸Ga generator) the samplewas incubated for 10 minutes at 90° C. or RT (control) and thentransferred to RT. 6 ml 0.1 M sodium citrate was added to neutralize thepH. The heat treated PEP08296 (0.8 and 4 nM) was injected at a constantflow-rate of 50 μl/min for 5 minutes, followed by dissociation in HBS-EPfor 15 minutes. The surfaces were regenerated with one injection of 25μl 10 mM HCl. The results were analyzed with BIAevaluation software (GEHealthcare). Curves of the blank surface were subtracted from the curvesof the ligand surfaces.

Determination of the Melting Temperature (Tm)

PEP08296 was dissolved in PBS to a final concentration of 0.5 mg/ml. PBSwith a pH of approximately 4.0 was prepared by adding 9.5 μl 100 mM HClto 100 μl PBS. Circular dichroism (CD) analysis was performed asdescribed in Example 3.

CD Analysis of Heat Stability

To investigate structural reversibility of PEP08296 after heattreatment, two CD spectra between 195 and 250 were recorded per sampleat 20° C. After the first spectrum, a VTM cycle with heating to 90° C.was run as described above followed by collection of the second CDspectrum between 195 and 250 nm at 20° C. In addition, PEP08296 wasincubated in PBS pH 4.0 buffer or PBS pH 7.2 buffer for 12 minutes at90° C. in a thermomixer (500 rpm, interval mixing 10 s on, 30 s off).After incubation, the samples were cooled on ice followed bycentrifugation at 13000 rpm for 1 minute, and a CD spectrum between 195and 250 nm was recorded at 20° C.

Results

Biosensor analysis was used to investigate if heat treatment incombination with low pH, i.e. common conditions needed for ⁶⁸Ga-labelingof polypeptide, would affect the capacity of PEP08296 to bind to HSA.FIG. 10 shows the result of this binding analysis performed with aBiacore 2000 instrument. Two different concentrations of PEP08296, 0.8nM and 4 nM, were injected over a surface with 724 RU of immobilizedhuman serum albumin. Heat treatment for 10 min at 90° C., pH 4.0,slightly reduced the binding capacity of PEP08296 to HSA, indicating apotential structural change of the molecule.

CD was used to further investigate the potential structural change ofthe molecule. Similar CD spectra before and after heating would prove asample to be structurally reversible. In the first experiment, thesamples were heated with a temperature gradient from 20° C. to 90° C.The CD spectra before and after heat treatment were similar in the Tmdetermination experiment with the typical minima at 207 and 221 nmindicating a-helicity, i.e. short time heating to 90° C. in either pH 4or pH 7.2 buffer had no effect on the structure of PEP08296.

However, pretreatment of PEP08296 for 12 minutes at 90° C. showed aslightly reduced alpha helix content of PEP08296 if incubated at pH 4.0,but no change in alpha helix content if incubated at pH 7.2. Typicaloverlays of two CD spectra before and after heating are shown in FIG.11.

The results from the melting temperature (Tm) determination aresummarized in Table 4.

TABLE 4 Tm of PEP08296 Designation Tm (° C.) PEP08296 at pH 7.2 59PEP08296 at pH 4.0 62

Example 11 Blood Pool Imaging Using a ⁶⁸Ga-Labeled Albumin BindingPolypeptide

In the experiments making up this example, whole body distribution of⁶⁸Ga-labeled PEP08296 (DOTA-conjugated PEP08185, SEQ ID NO:148) in ratswas followed by dynamic imaging over 1.5 hours. Due to the strongassociation between the labeled polypeptide and serum albumin, thelabeled polypeptide can be used for example to study blood pool andtissue permeability.

Material and Methods ⁶⁸Ga-Labeling of PEP08296

⁶⁸Ga was eluted as ⁶⁸GaCl₃ from the ⁶⁸Ge/⁶⁸Ga generator (Obninsk,Russia) with 0.1 M HCl, converted to ⁶⁸GaCl₄— with concentrated HCl,trapped on an anionic exchange column (Chromafix-HCO₃) and subsequentlyeluted with 18 MΩ water, as previously described (Velikyan et al (2008),Nucl Med Biol 35:529-536).

The labeling was performed essentially as described in Tolmachev et al.(EJNMMI 37:1356-1367, 2010). The concentrated ⁶⁸Ga-eluate (150-200 μl)was added to PEP08296 (≧100 μg in 0.2 M sodium acetate buffer pH 5.5)and the pH was adjusted to 3.5-4 using sodium acetate (1.25 M) or HCl(0.1 M). The labeling mixture was incubated at 90° C. for 15 min beforecooling, and the labeled protein was isolated by size exclusionpurification on a NAP-5 column eluted with physiologically bufferedsaline.

The radiochemical purity and identity of the ⁶⁸Ga-labeled protein wasassessed by radio-HPLC using UV (210 nm) and radioactivity detectors inseries and a Superdex Peptide 10/300 GL column (GE Healthcare) elutedwith physiologically buffered saline.

Small Animal PET

A rat (277 g) was anesthetized with isoflurane (initially 5%, then 2%blended with 7:3 air/O₂), controlled by an E-Z vaporizer using Microflexnon-rebreather masks from Euthanex Corporation, and was kept on aheating pad (37° C.) while lying within a microPET Focus120 system(Siemens, CTI Concorde Microsystems). ⁶⁸Ga-PEP08296, 33 MBq, wasdispensed in a syringe, diluted with saline to 0.5 ml and injected viathe tail vein. Data were acquired from the whole body by moving the bedin a constant bed motion protocol for 1.5 h. Data were processed withMicroPET Manager and corrected for randoms, dead time and decay. Imageswere reconstructed by standard 2D filtered back projection using a rampfilter and evaluated using Inveon Research Workplace (Siemens MedicalSolutions) software.

Results

Basic distribution patterns (FIG. 12) for PEP08296 were very similar tothat of albumin labeled with radioisotopes such as ⁶⁸Ga-DOTA, ⁶⁴Cu-DOTAand ¹¹C (see e.g. Hoffend et al (2005), Nucl Med Biol 32:287-292 and Luet al (2008), “[1-¹¹C]Butanol and [Methyl-¹¹C]Albumin for Blood Flow andBlood Pool Imaging”, poster at the Xlth Turku PET Symposium, 24-27 May2008). In brief, high radioactivity concentrations were observed inmajor blood vessels throughout the scan. Organs with large blood volumes(liver, spleen and kidney) were also clearly delineated, as was thecardiac blood pool radioactivity. Radioactivity in the urinary bladderincreased during the observation period, this observation of renalelimination being consistent with previous observations with labeledalbumin-based tracers and with that of the metabolism of albumin itself.

The general distribution pattern of radioactivity and very slow plasmaclearance after intravenous injection of ⁶⁸Ga-PEP08296 is consistentwith its expected very rapid and strong binding to albumin. Theseresults therefore support further applications of the radiotracer as anin vivo blood pool imaging agent for use with positron emissiontomography studies of tissue permeability, both during the developmentof disease and during therapeutic intervention.

Example 12 Solubility of an Albumin Binding Polypeptide

The solubility of PEP07986 (SEQ ID NO:163) in physiological buffer wasinvestigated by consecutive concentrations of the sample usingultrafiltration, followed by concentration measurement and investigationof aggregation status. Concentrations determined by direct absorbancereadings at 280 nm were consistent with concentrations determined by gelfiltration, showing a solubility of more than 42 mg/ml with noaggregates detected.

Material and Methods

Lyophilized PEP07986 was dissolved in NaPi buffer (20 mM sodiumphosphate, 150 mM sodium chloride, pH 7.2) at a concentration of 3mg/ml. Amicon Ultra centrifugal filter units, cut off of 3 kDa,(Millipore, Cat. No. UFC800324) were prerinsed with 2 ml NaPi buffer bycentrifugation at 4000 g for 20 min in a swinging bucket rotorcentrifuge (Multifuge, Heraeus). 1620 μl of 3 mg/ml PEP07986 was appliedto a first centrifugal filter unit and centrifugation was performed at4000 g, 20° C., for 7 min. A 25 μl sample was removed (UF sample 1) forfurther analysis and the rest of the sample was transferred to a secondcentrifugal filter unit. The centrifugation and sample removal wererepeated three times with spinning times of 8, 9 and 20 min respectively(UF sample 2, 3 and 4 respectively). Absorbance readings were performedusing a NanoDrop® ND-1000 Spectrophotometer and by diluting UF samples1-4 in NaPi buffer 2, 4, 6 and 12 times respectively. The concentrationswere calculated using the extinction coefficient 1 Abs 280=1.955 mg/ml.Gel filtration was performed on a 1100 HPLC system (AgilentTechnologies) using a Superdex75 10/300 GL column (GE Healthcare) whichhad been equilibrated in NaPi buffer. 10 μl of each UF sample wereapplied to the column; NaPi buffer was used as running buffer and theflow rate was 0.5 ml/min. A chromatogram of the molecular weightstandard ovalbumin (GE Healthcare), injected at a concentration of 5mg/ml was collected as well. Concentrations were determined byintegrating the area under the curve.

Results

Concentrations determined by direct absorbance readings at 280 nm andconcentrations determined by gel filtration are shown in Table 5. Thesolubility of PEP07986 (SEQ ID NO:163) is at least 42 mg/ml inphysiological buffer (20 mM sodium phosphate, 150 mM sodium chloride, pH7.2). No aggregates were detected by gel filtration, as shown by FIG.13.

TABLE 5 Concentrations determined after consecutive concentration ofPEP07986 (SEQ ID NO: 163) Concentrations (mg/ml) determined by SampleSpectrophotometer Gel filtration UF2 12.1 12.4 UF3 22.2 22.1 UF4 42.742.6

1. Albumin binding polypeptide comprising an amino acid sequenceselected from: i)  LAX₃AKX₆X₇ANX₁₀ ELDX₁₄YGVSDF YKRLIX₂₆KAKT VEGVEALKX₃₉X₄₀

wherein independently of each other X₃ is selected from E, S, Q and C;X₆ is selected from E, S and C; X₇ is selected from A and S; X₁₀ isselected from A, S and R; X₁₄ is selected from A, S, C and K; X₂₆ isselected from D and E; X₃₉ is selected from D and E; X₄₀ is selectedfrom A and E; X₄₃ is selected from A and K; X₄₄ is selected from A, Sand E; L in position 45 is present or absent; and P in position 46 ispresent or absent; and ii) an amino acid sequence which has at least 95%identity to the sequence defined in i).
 2. Albumin binding polypeptideaccording to claim 1, wherein X₆ is E.
 3. Albumin binding polypeptideaccording to claim 1, wherein X₃ is S.
 4. Albumin binding polypeptideaccording to claim 1, wherein X₃ is E.
 5. Albumin binding polypeptideaccording to claim 1, wherein X₇ is A.
 6. Albumin binding polypeptideaccording to claim 1, wherein X₁₄ is S.
 7. Albumin binding polypeptideaccording to claim 1, wherein X₁₄ is C.
 8. Albumin binding polypeptideaccording to claim 1, wherein X₁₀ is A.
 9. Albumin binding polypeptideaccording to claim 1, wherein X₁₀ is S.
 10. Albumin binding polypeptideaccording to claim 1, wherein X₂₆ is D.
 11. Albumin binding polypeptideaccording to claim 1, wherein X₂₆ is E.
 12. Albumin binding polypeptideaccording to claim 1, wherein X₃₉ is D.
 13. Albumin binding polypeptideaccording to claim 1, wherein X₃₉ is E.
 14. Albumin binding polypeptideaccording to claim 1, wherein X₄₀ is A.
 15. Albumin binding polypeptideaccording to claim 1, wherein X₄₃ is A.
 16. Albumin binding polypeptideaccording to claim 1, wherein X₄₄ is A.
 17. Albumin binding polypeptideaccording to claim 1, wherein X₄₄ is S.
 18. Albumin binding polypeptideaccording to claim 1, wherein L in position 45 is present.
 19. Albuminbinding polypeptide according to claim 1, wherein P in position 46 ispresent.
 20. Albumin binding polypeptide according to claim 1, whichbinds to albumin such that the k_(off) value of the interaction is atmost 5×10⁻⁵ s⁻¹.
 21. Albumin binding polypeptide according to claim 20,which binds to albumin such that the k_(off) value of the interaction isat most 5×10⁻⁶ s⁻¹.
 22. Albumin binding polypeptide according to claim1, whose amino acid sequence is selected from any one of SEQ ID NO:1-144and SEQ ID NO:164-203.
 23. Albumin binding polypeptide according toclaim 22, whose amino acid sequence is selected from any one of SEQ IDNO:4-5, SEQ ID NO:7-8, SEQ ID NO:10-11, SEQ ID NO:13-14, SEQ IDNO:16-17, SEQ ID NO:19-20, SEQ ID NO:22-23, SEQ ID NO:25-26, SEQ IDNO:28-29, SEQ ID NO:31-32, SEQ ID NO:34-35, SEQ ID NO:37-38, SEQ IDNO:41-42, SEQ ID NO:49-50, SEQ ID NO:164-170 and SEQ ID NO:192-203. 24.Albumin binding polypeptide according to claim 22, whose amino acidsequence is selected from any one of SEQ ID NO:1-144.
 25. Albuminbinding polypeptide according to claim 24, whose amino acid sequence isselected from any one of SEQ ID NO:4-5, SEQ ID NO:7-8, SEQ ID NO:10-11,SEQ ID NO:13-14, SEQ ID NO:16-17, SEQ ID NO:19-20, SEQ ID NO:22-23, SEQID NO:25-26, SEQ ID NO:28-29, SEQ ID NO:31-32, SEQ ID NO:34-35, SEQ IDNO:37-38, SEQ ID NO:41-42 and SEQ ID NO:49-50.
 26. Albumin bindingpolypeptide according to claim 1, further comprising one or moreadditional amino acid residues positioned at the N- and/or theC-terminal of the sequence defined in i).
 27. Albumin bindingpolypeptide according to claim 26, in which the additional amino acidscomprise at least one serine residue at the N-terminal of thepolypeptide.
 28. Albumin binding polypeptide according to claim 26, inwhich the additional amino acids comprise a glycine residue at theN-terminal of the polypeptide.
 29. Albumin binding polypeptide accordingto claim 26, in which the additional amino acids comprise a cysteineresidue at the N-terminal of the polypeptide.
 30. Albumin bindingpolypeptide according to claim 26, in which the additional amino acidscomprise a lysine residue at the C-terminal of the polypeptide. 31.Albumin binding polypeptide according to claim 26, in which theadditional amino acids comprise a glycine residue at the C-terminal ofthe polypeptide.
 32. Albumin binding polypeptide according to claim 26,in which the additional amino acids comprise a cysteine residue at theC-terminal of the polypeptide.
 33. Albumin binding polypeptide accordingto claim 26, whose amino acid sequence is selected from any one of SEQID NO:145-150 and SEQ ID NO:162-163.
 34. Albumin binding polypeptideaccording to claim 1, comprising no more than two cysteine residues. 35.Albumin binding polypeptide according to claim 34, comprising no morethan one cysteine residue.
 36. Albumin binding polypeptide according toclaim 1, which binds to human serum albumin.
 37. Fusion protein orconjugate comprising i) a first moiety consisting of an albumin bindingpolypeptide according to claim 1; and ii) a second moiety consisting ofa polypeptide having a desired biological activity.
 38. Fusion proteinor conjugate according to claim 37, in which the second moiety having adesired biological activity is a therapeutically active polypeptide. 39.Fusion protein or conjugate according to claim 38, in which the secondmoiety having a desired biological activity is selected from the groupconsisting of human endogenous enzymes, hormones, growth factors,chemokines, cytokines and lymphokines.
 40. Fusion protein or conjugateaccording to claim 39, in which the second moiety is selected from thegroup consisting of IL-2, GLP-1, BNP, IL-1-RA, KGF, Stemgen®, GH, G-CSF,CTLA-4, myostatin, Factor VII, Factor VIII and Factor IX.
 41. Fusionprotein or conjugate according to claim 38, in which the second moietyhaving a desired biological activity is a non-human biologically activeprotein, selected from the group consisting of bacterial toxins, enzymesand activating proteins.
 42. Fusion protein or conjugate according toclaim 37, in which the second moiety having a desired biologicalactivity is a binding polypeptide capable of selective interaction witha target molecule.
 43. Fusion protein or conjugate according to claim42, in which the binding polypeptide is selected from the groupconsisting of antibodies and fragments and domains thereof substantiallyretaining antibody binding activity; microbodies, maxybodies, avimersand other small disulfide-bonded proteins; and binding proteins derivedfrom a scaffold selected from the group consisting of staphylococcalprotein A and domains thereof, other three helix domains, lipocalins,ankyrin repeat domains, cellulose binding domains, [gamma] crystallines,green fluorescent protein, human cytotoxic T lymphocyte-associatedantigen 4, protease inhibitors such as Kunitz domains, PDZ domains, SH3domains, peptide aptamers, staphylococcal nuclease, tendamistats,fibronectin type III domain, transferrin, zinc fingers and conotoxins.44. Fusion protein or conjugate according to claim 43, in which saidtarget molecule is selected from the group consisting of [Alpha] [beta]peptide of Alzheimer's disease; other disease-associated amyloidpeptides; toxins, such as bacterial toxins and snake venoms; bloodclotting factors, such as von Willebrand factor; interleukins, such asIL-13; myostatin; pro-inflammatory factors, such as TNF-a, TNF-areceptor, IL-1, IL-23 and IL-8; complement factors, such as C3 and C5;hypersensitivity mediators, such as histamine and IgE; tumor-relatedantigens, such as CD19, CD20, CD22, CD30, CD33, CD40, CD52, CD70, cMet,HER1, HER2, HER3, HER4, CAIX, CEA, IL-2 receptor, MUC1, PSMA, TAG-72,and other biological molecules such as G-CSF, GM-CSF, GH, insulin andsomatostatin.
 45. Fusion protein or conjugate according to claim 37,comprising a further moiety consisting of a polypeptide having afurther, desired biological activity, which may be the same as ordifferent from that of the second moiety.
 46. Fusion protein orconjugate according to claim 45, wherein the second moiety is as definedin claim 38, and the further moiety is as defined in claim
 42. 47.Conjugate according to claim 37, in which the second moiety isconjugated to an albumin binding polypeptide according to claim 1 viathe thiol group of any cysteine residue present at position X₁₄ of thepolypeptide.
 48. Albumin binding polypeptide, fusion protein orconjugate according to claim 1, further comprising a cytotoxic agent.49. Albumin binding polypeptide, fusion protein or conjugate accordingto claim 48, wherein the cytotoxic agent is selected from calicheamycin,auristatin, doxorubicin, maytansinoid, taxol, ecteinascidin,geldanamycin, methotrexate and their derivatives, and combinationsthereof.
 50. Albumin binding polypeptide, fusion protein or conjugateaccording to claim 1 further comprising a label.
 51. Albumin bindingpolypeptide, fusion protein or conjugate according to claim 50, in whichsaid label is selected from the group consisting of fluorescent dyes andmetals, chromophoric dyes, chemiluminescent compounds and bioluminescentproteins, enzymes, radionuclides and particles.
 52. Albumin bindingpolypeptide, fusion protein or conjugate according to claim 51,comprising a chelating environment provided by apolyaminopolycarboxylate chelator conjugated to the albumin bindingpolypeptide via a thiol group of a cysteine residue or an amine group ofa lysine residue.
 53. Albumin binding polypeptide, fusion protein orconjugate according to claim 52, wherein the polyaminopolycarboxylatechelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ora derivative thereof.
 54. Albumin binding polypeptide, fusion protein orconjugate according to claim 53, wherein the1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid derivative is1,4,7,10-tetraazacyclododecane-1,4,7-tris-aceticacid-10-maleimidoethylacetamide.
 55. Albumin binding polypeptide, fusionprotein or conjugate according to claim 52, wherein thepolyaminopolycarboxylate chelator is diethylenetriaminepentaacetic acidor derivatives thereof.
 56. Polynucleotide encoding an albumin bindingpolypeptide or a fusion protein according to claim
 1. 57. Method ofproducing a polypeptide according to claim 1, comprising expressing apolynucleotide according to claim
 56. 58. Expression vector comprising apolynucleotide according to claim
 56. 59. Host cell comprising anexpression vector according to claim
 58. 60. Method of producing apolypeptide according to claim 1 by non-biological peptide synthesisusing amino acids and/or amino acid derivatives having protectedreactive side-chains, the non-biological peptide synthesis comprisingstep-wise coupling of the amino acids and/or the amino acid derivativesto form a polypeptide according to claim 1 having protected reactiveside-chains, removal of the protecting groups from the reactiveside-chains of the polypeptide, and folding of the polypeptide inaqueous solution.
 61. Method of producing a conjugate according to claim37, comprising producing an albumin binding polypeptide by the methodaccording to claim 60, and conjugating the produced albumin bindingpolypeptide with a second and/or further moiety as defined in claim 38.62-64. (canceled)
 65. Composition, comprising a compound which per sehas a solubility in water of no more than 100 g/ml; coupled to analbumin binding polypeptide, a fusion protein or conjugate according toclaim 1, wherein the compound and the albumin binding polypeptide,fusion protein or conjugate are covalently coupled.
 66. Compositionaccording to claim 65, wherein said solubility is no more than 10 g/ml.67. Composition according to claim 66, wherein said solubility is nomore than 1 g/ml.
 68. Composition according to claim 65, wherein saidcompound is a pharmaceutically active compound.
 69. Compositionaccording to claim 68, wherein said pharmaceutically active compound isa cytotoxic agent.
 70. Composition according to claim 69, wherein saidcytotoxic agent is as defined in claim
 49. 71. Composition according toclaim 69, wherein said cytotoxic agent is a synthetic chemotoxin notderived from a naturally occurring compound.
 72. Composition accordingto claim 65, further comprising a binding polypeptide having a bindingaffinity for a clinically relevant target.
 73. Composition according toclaim 72, wherein said binding polypeptide is as defined in claim 43.74. Composition according to claim 65, further comprising human serumalbumin. 75-76. (canceled)
 77. Method of preparation of a compositionaccording to claim 65, comprising a) providing a compound which per sehas a solubility in water of no more than 100 μg/ml; and b) covalentlycoupling the compound to an albumin binding polypeptide, fusion proteinor conjugate according to claim 1, thus forming a composition comprisinga covalent complex of compound and an albumin binding polypeptide,fusion protein or conjugate.
 78. Method according to claim 77, furthercomprising c) mixing said complex of compound and albumin bindingpolypeptide, fusion protein or conjugate with albumin, thus forming acomposition comprising a non-covalent complex of i) the covalent complexof compound and albumin binding polypeptide, fusion protein or conjugateand ii) albumin.
 79. Method according to claim 78, further comprisinglyophilizing said composition comprising the non-covalent complex toobtain a lyophilized composition.
 80. Method of increasing the aqueoussolubility of a compound, comprising providing a compound which per sehas a solubility in water of no more than 100 μg/ml; covalently couplingthe compound to an albumin binding polypeptide, fusion protein orconjugate according to claim 1, thus forming a covalent complex ofcompound and albumin binding polypeptide; and mixing said complex ofcompound and albumin binding polypeptide, fusion protein or conjugatewith albumin under conditions that promote the non-covalent associationof the albumin binding polypeptide with human serum albumin; whereby thesolubility in water of the compound in said complex is greater than thesolubility in water of the compound per se.
 81. Method according toclaim 80, wherein said solubility is as defined in claim
 66. 82. Methodaccording to claim 80, wherein said compound is as defined in claim 68.83. Method according to claim 80, wherein said complex further comprisesa polypeptide having a binding affinity for a clinically relevanttarget.
 84. Method according to claim 83, wherein said bindingpolypeptide is as defined in claim 43.